Intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome 1 (PRRS1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells in the presence of maternally derived antibodies

Intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome 1 (PRRS1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells in the presence of maternally derived antibodies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome 1 (PRRS1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells in the presence of maternally derived antibodies Laia Aguirre, Yanli Li, Massimiliano Baratelli, Gerard Martín-Valls, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1857671/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The purpose of this study was to compare the immune response generated by the intramuscular and the intradermal vaccination route against the porcine reproductive and respiratory syndrome virus (PRRSV). Piglets from a seronegative and a seropositive farm were selected (n = 28 piglets per farm), and each group was divided into two groups and vaccinated at weaning with modified live vaccine Unistrain® PRRS (Laboratorios Hipra Amer, Spain) by the intramuscular or the intradermic route. For the following 6 weeks, animals were weekly bled to assess the humoral response by PRRSV-specific antibody ELISA and viral neutralisation test. At 0-, 3-, 4- and 6 weeks post-vaccination, peripheral mononuclear blood cells (PBMC) from eight animals per group were recovered to analyse cellular response by IFN-γ ELISPOT and lymphoproliferation. Serum IL-12 was also quantified by ELISA. Results showed no significant differences between treatments for any of the parameters studied. However, pigs from the seronegative origin had higher dispersion in S/P ratios by ELISA (Levene’s test for homogeneity of variances, p < 0.05). At 3 weeks after vaccination, 6/27 (22.22%) animals from negative origin had not seroconverted. Also, it was 10 times more probable for them to have high levels of IL-12 a week after vaccination than for animals of seropositive origin. These results indicate that the intradermal route induces an immune response equivalent to the classical intramuscular route even in presence of maternal immunity, which in this study has proven to facilitate seroconversion after vaccination. Porcine reproductive and respiratory syndrome virus vaccine Intramuscular Intradermal Maternally derived antibodies. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The porcine reproductive and respiratory syndrome (PRRS) is one of the main causes of economic loss in swine worldwide. Control of the infection is usually done by a combination of virus monitoring, biosecurity restrictions, herd management measures, and vaccination. Although vaccination most often targets sows, piglet vaccination is increasingly being considered to protect weaners in farms that have endemic problems in nurseries. Vaccination of piglets against PRRS virus (PRRSV) is usually performed during lactation or immediately after weaning. In endemically infected farms, most piglets present high levels of maternally derived antibodies (MDA) against PRRSV which may interfere with the development of an active immunity after vaccination [ 1 , 2 ]. The mechanisms of blocking by MDA are diverse (revised by Niewiesk [ 3 ]) and most often do not involve neutralization of the antigen but the interaction of MDA with B-cell receptors. Different strategies can be used to avoid the eventual interference caused by maternal antibodies on vaccine immunisation. For instance, one common approach is to administer repeated doses of the vaccines (commonly two doses separated by a 3-4-week period) close to the age at which MDA are expected to wane to compensate for the eventual detrimental effect of the MDA on the first dose. Another option is to deliver the antigen to anatomical sites where the concentration of MDA is lower than the concentration in the muscle so there is less chance of interference with the vaccination. The intradermal route has several advantages such as the reduction of the antigen needed to induce an immune response [ 4 ] and the potential of targeting a higher diversity of dendritic cells (reviewed by Combadiere and Liard [ 5 ]). In PRRS, the efficacy of vaccination by intradermal route has been proven to be similar to the intramuscular route [ 6 – 9 ] when pigs were not having specific MDA. However, much less is known about the efficacy of the intradermal route in the presence of MDA. The main objective of the present study was to compare the immune responses generated by a PRRSV live attenuated vaccine administered by either the intradermal or the intramuscular routes in piglets with high levels of specific MDA or in seronegative piglets. Results 1. Clinical follow-up No adverse reactions to vaccination were recorded. Two animals died during the study, one because of an internal haemorrhage (probably because of a fight) and the other one because of diarrhoea. Two animals were euthanised because of the development of bacterial meningitis. One additional animal was withdrawn from the study on arrival at the experimental facilities because of a lameness produced during the load and transport. No other incidents were observed in the rest of the animals during the 42-day follow-up. 2. Vaccine-induced viremia Figure 1 shows the evolution of vaccine-induced viremia in the different groups. After vaccination animals were tested weekly to evaluate the presence of PRRS virus in blood. In all groups, vaccination produced a viremia at 7 days post-vaccination (dpv) (from 50–91% of the vaccinated animals, depending on the group). After 21 dpv the proportion of viremic animals dropped significantly and after 35 dpv, only 1 or 2 animals/group were positive. No significant differences between groups were observed regarding the proportion of positive animals. Regarding Ct-values, significant differences were observed at 7 dpv between ID-POS and the IM-NEG with lower Ct values for the latter (Supplementary material S1). 3. Development of antibodies The cohort of pigs tested in the seropositive herd (n = 132) showed S/P values (ELISA) ranging between 0.44 and 2.24 (Supplementary material S2). The 28 selected animals had S/P values from 1.45 to 2.24. Those S/P values were in the quartile 75% of the distribution. At 4 weeks of age, when the vaccination was performed, animals in subgroup IM-POS had an S/P of 1.22 ± 0.28 versus 1.16 ± 0.23 for ID-POS (non-significant differences). As expected, animals in group NEG were seronegative at the moment of vaccination. Figure 2 shows the evolution of average S/P values per group after vaccination. Vaccination produced a clear seroconversion in the seronegative animals (group NEG) that was noticeable by 14 dpv. Average S/P values increased until 35 dpv in the IM-NEG group, and until 42 dpv in the ID-NEG group. In the POS groups seroconversion was also observed, with increasing S/P ratios until day 28 dpv. It is worth noting that the ID-POS group presented the highest average S/P value at the end of the study, significantly different from that of the ID-NEG group. Differences between POS and NEG IM-vaccinated animals were non-significant. Sera obtained on the day of vaccination could neutralise the vaccine used in sows (titres of 2.75 ± 1.42 log 2 and 3.2 ± 2.1 log2 in IM and ID, respectively, non-significant) but they were devoid of neutralisation capacity against the vaccine administered to piglets. The development of neutralising antibody titres induced by the administration of the vaccine was firstly detected at 28 dpv. At 35 dpv, significant differences were noticed between the groups that were vaccinated in the presence of MDA and the seronegative ones (Fig. 3, p < 0.05). Thus, the animals of the seropositive origin presented higher titres compared to animals from the negative origin being IM or ID administration results equivalent. 4. Lymphoproliferation PRRS-specific lymphocyte proliferation was clear by 21 dpv in some animals, which was more evident at 28 dpv and present in most animals at 35 dpv. Some differences in the proliferative responses were noticed at 35 dpv favouring the IM-NEG group (Kruskal-Wallis, p < 0.05), but they disappeared at 42 dpv (Fig. 4 ). 5. IFN-γ secreting cells ELISPOT results were negative for all the animals at 0 dpv. Virus-specific responses were detected at 21 dpv in several animals from different groups and peaked at 42 dpv, with no significant differences between groups at any age (Fig. 5 ). 6. Cytokine production IFN-α. Levels of IFN-α in serum at 7 dpv ranged between 300 and 700 pg/mL, with no significant differences between groups (IM-NEG: 435 ± 83 pg/ml, ID-NEG: 420 ± 79; ID-POS: 454 ± 86; ID-POS: 481 ± 86). IL-10. At 7 dpv, levels of IL-10 were negative or very low (< 64 pg/ml) in all animals with no differences between groups. At 14 dpv values higher than 100 pg/ml of IL10 could be detected only in sera of NEG animals, 4 in the ID subgroup (164-1,849 pg/ml) and 2 in the IM subgroup (106–553 pg/ml). Of these, at 21 dpv 2 animals still showed elevated IL10 levels in the ID-NEG group (249–505 pg/ml) and one in the IM-NEG group (273 pg/ml). In culture supernatants, IL-10 concentration was very low and with no significant differences. IL-12. At 7 dpv it was 10 times more likely to be IL-12 positive (9/28 vs. 1/23; p = 0.033) for animals of the seronegative origin compared to the seropositive ones. At 14 dpv, all animals but three showed high levels of IL-12 in serum (195.1-3,821.6 pg/ml). Interestingly, of the 10 animals having the highest IL-12 levels at 14 dpv, 6 were the ones having elevated IL-10 at the same sampling time. Discussion Interference of MDA with vaccination is a major concern when young piglets are to be vaccinated. The mechanisms by which that interference occurs are not fully elucidated. However, there is strong evidence favouring the hypothesis that blocking by MDA may involve the inhibition of B-cell responses by the cross-linking between B cell receptor with the Fcγ-receptor IIB by a vaccine–antibody complex, regardless of the neutralising capacity of the antibodies involved [ 3 , 10 , 11 ]. The intradermal administration of vaccines is an increasingly interesting alternative for vaccination, being less invasive and more respectful of animal welfare [ 12 ]. Interference with MDA could also be diminished by this vaccination route, as shown for the human poliovirus vaccine administered to children [ 13 ]. The skin has a rich diversity of antigen-presenting cells that are highly efficient for capturing and transporting antigens to the draining lymph nodes. Of special interest are Langerhans cells that are particularly able to induce cytotoxic T cells [ 14 ] but are less efficient in inducing responses from B cells. On the other hand, in humans, CD14 + dermal dendritic cells are especially able to prime naïve B cells. This diversity of antigen-presenting cells in the skin can be advantageous for achieving effective immune responses after vaccination even when reduced amounts of antigen are available. Several studies proved that reduced doses of dermally-delivered antigens are effective for immunizing children with MDA against poliovirus [ 13 , 15 ]. In the case of PRRSV, the intradermal administration of MLV PRRSV vaccines has proven to be as effective as the IM administration in naïve MDA-free pigs [ 6 – 9 , 16 ]. The vaccine tested in the present study showed that both routes were equally effective in developing immunity against PRRS in absence of MDA, which is in line with what showed by the above authors; moreover, the vaccine was demonstrated to be able to produce similar levels of immunity by means both routes even in presence of high MDA levels. It is worth mentioning here that, in PRRSV endemic farms, circulation of the virus often starts in the farrowing units because of the existence of vertical transmission from sows to newborns, or very soon after weaning since MDA wane between 4 and 5 weeks of age (reviewed by Pileri and Mateu [ 17 ]). Given the fast spread of the virus in naïve populations, vaccination of piglets must be administered at weaning or even before, implying that most vaccinated piglets will have MDA. Since the neutralising antibodies against PRRSV usually have a narrow breadth of neutralisation, the capability of MDA to neutralise a particular vaccine virus will depend on what vaccine was used in the sows and on their previous contact with other PRRSV strains. The most remarkable results were on animals of seropositive origin, which presented a stronger humoral response despite having high levels of MDA. This was clear for both S/P values (particularly for ID-POS) and virus neutralising antibodies (NA) titres. It is worth noting here that the scenario selected for the study was that where seropositive piglets did not have NA against the vaccine virus. Since NA in PRRS usually have a narrow range of neutralisation [ 18 ], this scenario would account for most situations, including the vaccination of the offspring of sows vaccinated with a different vaccine, or simply, of seropositive unvaccinated sows. Although NA may have a role in blocking the replication of the MLV virus, it is known that vaccine blocking by the MDA is not necessarily related to the presence of NA [ 3 ]. This phenomenon of increased response to vaccination in individuals with sub-neutralising levels of antibodies has been proven in humans that were vaccinated with an experimental attenuated vaccine against the dengue virus [ 19 , 20 ]. In that case, the greater immune response in individuals with antibodies was attributed to increased infection of macrophages mediated by virus-antibody complexes. In the present case, Ct values of POS animals at 7 dpv were similar or lower to those of NEG animals. However, it is worth noting that there were more PCR-positive pigs at 7 dpv in the POS (IM and ID) compared to the NEG groups. The design could have certainly overlooked the case of animals with high homologous NA titres. Renson et al. [ 1 ] showed that homologous neutralising MDA titres of about 1:10 may produce some interference with the development of immunity in piglets. The interference of MDA antibodies resulting from vaccination with different vaccines should be evaluated in the future. Regarding the cell-mediated responses, the presence of MDA did not significantly interfere with the development of proliferative or IFN-γ responses. In other studies, it has been shown that vaccine blocking by MDA usually affects the development of humoral responses but not the development of cell-mediated immunity [ 21 , 22 ]. In the present case, the development of vaccine-induced viremia was similar in all groups following a pattern described in other previous works [ 1 , 23 ]. As shown before, some animals did not develop detectable viremia while others persisted as positive for several weeks. One possible element involved in this different behaviour could have been the IFN-α response. Renson et al. [ 1 ] reported a potential correlation of this phenomenon with decreased response to the vaccine. This was not our case since all animals had comparable levels of this cytokine in serum. Interestingly, the results showed that seronegative animals had a higher probability of having high levels of IL-12 in serum after vaccination than seropositive ones. This suggests a differential targeting of TLRs in animals with or without antibodies against the vaccine virus. Most of the animals with the highest IL-12 levels in serum were the ones with high IL-10 levels. A possible explanation could be in the homeostatic action of IL-10 o counterbalance excessive IL-12 production. It is known that IL-10 is a potent regulator of IL-12 transcription [ 24 ]. These results raise questions concerning the role of MDA in PRRS vaccination. The present results would indicate that in cases where the MDA were subneutralising (i.e., heterologous vaccines in sows and piglets), humoral immunity induced by MLV vaccination could even be enhanced. Further research should be done in this area to figure out what were the mechanisms leading to this difference including the possible enhancement of the vaccine virus replication and also whether vaccine-antibody complexes result in different stimulation of antigen-presenting cells. Conclusion In conclusion, intradermal and intramuscular vaccinations have been proven as equivalent administration routes for the immunity parameters evaluated in this experimental study. The presence of non-neutralising antibodies at the time of vaccination resulted in enhanced humoral response without being detrimental to the development of cell-mediated responses. Material And Methods 1. Animals and experimental design The study was approved by the Committee for Ethics in Human and Animal Experimentation of the Universitat Autònoma de Barcelona (number CEEAH 5357) and by the Generalitat de Catalunya (FUE-2020-01836411). Pigs were recruited from two different herds; in the first, high levels of PRRSV-specific MDA were detected (group POS) whereas in the second no PRRSV antibodies were detected (group NEG). Group POS were DanBred x Pietrain crossbred animals. The farm was positive stable with vaccination according to the revised AASV scheme of classification as PRRSV was not detected at weaning [ 25 ]. At two weeks of age, 132 piglets were randomly selected from the same farrowing batch and bled to determine the levels of MDA by ELISA (PRRS X3 Ab, Idexx). The absence of PRRSV circulation in the group was confirmed by RT-qPCR (VetMax PRRS EU&NA 2.0 RT-qPCR, ThermoFisher, Madrid, Spain). The 28 animals with the highest S/P ratios as determined by the test were selected for the experiment. Group NEG animals (n = 28) were Duroc x Landrace pigs obtained from a historically PRRSV-free farm and its status was confirmed as well by ELISA. In both cases, animals were weaned at 3 weeks of age and transported to the experimental facilities located in the UAB. There, they were ear-tagged and randomly divided into two subgroups that were housed in physically separated boxes. One subgroup was assigned the intramuscular (IM) vaccination and the other was assigned the intradermal (ID) vaccination. Thus, the final design contained 4 subgroups: IM-POS, IM-NEG, ID-POS, and ID-NEG according to the serological status and the route of administration of the vaccine. Pigs were left to acclimate for a week before the administration of the vaccine. At 4 weeks of age, animals were bled and the PRRSV1 vaccine was administered (Unistrain® PRRS, Laboratorios Hipra). The IM vaccination was performed by the injection of 2 ml of the vaccine in the neck muscles; the ID vaccination was performed by delivering 0.2 ml of the vaccine in the neck using a needle-free device (Hipradermic® 3.0). Each dose of the vaccine contained 1x10 4.3 TCID 50 as titrated in MARC-145 cells. 2. Sampling and sample processing Animals were monitored for 42 days after vaccination. Blood samples were obtained weekly from all animals to determine the development of PRRSV-specific antibodies. Heparinized blood samples were additionally taken from 8 animals/group at 0-, 21-, 28-, and 42 dpv. The heparinized samples were used to obtain peripheral mononuclear blood cells (PBMC) by gradient density centrifugation using Histopaque-1077® (Sigma-Aldrich) and Sepmate™ tubes (Stemcell Technologies, Saint-Égrève, France). The resulting PBMC were frozen in Cryostor® CS10 (Merck) and stored in liquid nitrogen until further analysis. PBMC were recovered as described elsewhere [ 26 ] and viability was checked by trypan blue staining (0.4%). Only samples with > 90% viability were used. 3. Serological analysis Serum samples were tested for the presence of PRRSV specific antibodies by HerdCheck® PRRS X3 Ab test Idexx ELISA. Viral neutralisation tests were performed in sera from 0-, 21-, 28-, 35-, and 42 dpv using the vaccine strain. For group POS animals, samples of 0 dpv were also tested against the vaccine strain used for the sows in the origin farm (Porcilis™ PRRS; MSD Animal Health). The neutralisation tests were performed following the procedure described by Yoon et al. (27) with minor modifications. Briefly, sera were inactivated at 56°C for 30 minutes and diluted from 1:2 to 1:256 in Minimum Essential Medium with non-essential amino acids, sodium pyruvate, 100 IU/mL penicillin and 100 µg/mL streptomycin. Equal volumes (100 µl:100 µl) of the diluted serum and the vaccine strain adjusted at 2,000 TCID 50 /mL were mixed and incubated overnight at 4°C. Then, 100 µL of the serum-virus mixture was transferred to MARC-145 monolayers in 96 well-plates. Negative (only medium) and virus controls (virus at working dilution and at 200, 20, and 2 TCID 50 /mL) were also included in the plate, as well as a negative and positive serum (post-vaccination serum of a sow) as controls. Plates were read after 6 days of incubation. The neutralisation titres were assessed by the development of the cytopathic effect and confirmed by immunofluorescence using the monoclonal antibody 1CH5 (Eurofins, Madrid). The neutralisation titre was the log 2 of the reciprocal of the highest dilution without cytopathic effect and without significant fluorescence. 4. Quantification of PRRSV RNA Quantitative PCR for the quantification of PRRSV1 in serum was performed as previously reported [ 28 ]. 5. Lymphoproliferation Isolated PBMC were stained with CellTrace™ Violet (ThermoFisher) following the manufacturer's instructions. Briefly, 1x10 5 PBMC were dispensed on 96-well round-bottomed plates and the vaccine strain was added at a multiplicity of infection (MOI) of 0.1 (cultures were done in triplicate). In parallel, phytohemagglutinin-stimulated cultures (PHA, 10 µg/mL) or mock-stimulated cultures (RPMI medium) were used as positive and negative controls respectively. Plates were incubated for 5 days at 37°C in a 5% CO 2 atmosphere. Then, cell cultures were recovered and centrifuged at 300 g for 10 min. The resulting pellet was resuspended in PBS and analysed in a Cytoflex flow cytometer (Beckman Coulter, CA, USA). Flow cytometer files were analysed using the FCS Express Flow Cytometry 6 (de Novo Software). For each sample, the PRRSV-specific proliferation was calculated as follows: percentage of cell proliferation in the PRRSV stimulated cultures - percentage of cell proliferation in the negative control. A relative proliferation index was also calculated as the proportion of cell proliferation in PRRSV-stimulated culture versus the proportion of cell proliferation in the mock-stimulated cultures. 6. IFN-γ ELISPOT IFN-γ ELISPOT was performed as described by Zuckerman et al. [ 29 ] with minor modifications. Briefly, Costar 3590 plates (Corning) were coated with 50 µL of the monoclonal antibody P2G10 against porcine IFN-γ (ref. 559961; BD Pharmingen, NJ, USA) at 5 µg/mL diluted in carbonate-bicarbonate buffer (0.15M pH 9.5). After overnight incubation at 4°C, plates were washed five times with PBS and blocked for 1 hour with RPMI containing 10% foetal calf serum. Following the removal of the blocking solution, 50 µL/well of PBMC were dispensed at 1x10 5 PBMC/well (negative control and virus stimulated wells) and 5x10 4 PBMC/well (PHA-stimulated wells). PRRSV-stimulated wells were added to the vaccine strain at a MOI of 0.1. Mock-stimulated cultures (culture medium) and PHA-stimulated wells (10 µg/mL) were added as negative and positive controls, respectively. Plates were incubated overnight at 37°C with 5% CO 2 . After washing, the biotinylated detection antibody P2C11 (ref. 559958; BD Pharmingen) was added at 0.5 µg/mL diluted in PBS with 0.4% bovine serum albumin. After a 1 hour incubation at 37°C, 50 µL of streptavidin-peroxidase was added at 0.5 µg/mL (ThermoFisher) and the reaction was developed by adding insoluble TMB (50 µL/well). Spots were counted in a Leica stereoscope after a 10-minute incubation in the dark. Samples were tested in triplicate. The frequencies of responding cells were calculated by subtracting the counts in the mock-stimulated wells from the counts obtained in virus-stimulated wells for each sample. Results were expressed as the number of responding cells per 1x10 6 PBMC. 7. Cytokine ELISA in culture supernatant and serum ELISA for cytokines IL-10, IL-12 and IFN-α were performed in PBMC culture supernatants and/or serum. Cell culture supernatants were obtained from PBMC cultures produced in the same conditions as the ELISPOT. Supernatants were collected after overnight incubation of the PBMC and frozen at − 80°C until further analysis. Details of the samples analysed are shown in Table 1. Antibody pairs and kits used are shown in supplementary materials (S3). Optimal work concentrations of each antibody were previously determined using the reference standards provided by each kit. Cytokine concentrations in the supernatants were estimated by calculating a regression formula obtained from the results produced by a serial dilution of the standard provided by the kit. Declarations Ethics approval The study protocol was approved by the Committee for Ethics in Human and Animal Experimentation of the Universitat Autònoma de Barcelona (number CEEAH 5357) and by the Generalitat de Catalunya (FUE-2020-01836411). Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests Massimiliano Baratelli and Joel Miranda are employees of Laboratorios Hipra Funding This study was funded by Laboratorios Hipra, S. 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Martínez E, Riera P, Sitjà M, Fang Y, Oliveira S, Maldonado J. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green. Res Vet Sci. 2008;85:184–93. Zuckermann FA, Husmann RJ, Schwartz R, Brandt J, Mateu De Antonio E, Martin S. Interleukin-12 enhances the virus-specific interferon gamma response of pigs to an inactivated pseudorabies virus vaccine. Vet Immunol Immunopathol. 1998;63:57–67. Additional Declarations Competing interest reported. Massimiliano Baratelli and Joel Miranda are employees of Laboratorios Hipra. Supplementary Files S1.png Supplementary material S1. Average Ct values for the vaccine-induced viremia a 7dpv. The graph depicts the average Ct-value of PCR-positive animals at 7 dpv (plus maximum, minimum, 25% and 75% quartiles). Different superscript letters indicated significant differences (p<0.05, Kruskal-Wallis test). S2.png Supplementary material S2. Distribution of S/P ratios at the moment of the selection of seropositive animals. The graph depicts the distribution of S/P ratios for the group of animals sampled at 2 weeks of age for the selection of POS individuals. SupplementarymaterialS3.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 12 Sep, 2022 Reviews received at journal 09 Sep, 2022 Reviews received at journal 22 Aug, 2022 Reviewers agreed at journal 08 Aug, 2022 Reviewers invited by journal 08 Aug, 2022 Editor assigned by journal 20 Jul, 2022 Submission checks completed at journal 20 Jul, 2022 First submitted to journal 14 Jul, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1857671","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":122669567,"identity":"f17f4875-be78-4ae6-893f-529b456cd579","order_by":0,"name":"Laia Aguirre","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACPgbGBjCDDUR8YGBIIKiFDVkL4wyIFqgQTi1IgJmHKC3shxs/MNTUJvZJn3342LbNLo+B/fDzB3i18CQ2SzAcO57YxpdubJzbllzMwJNmSMBhiQ0SDGzHEtt42Nikc9uYQVwCWvgfNv9g+AfWwv7bsq0eqIX9I34tEoltEoxtNWBbmBnbDgO18BCwReJhm0Vi3wFjoBZmyZ5zx4vZeHIKZ+DTws+f/vjGh291svN72Bg//CirzuNnP77hAz4tYJDAcBjJXoLKIaCOSHWjYBSMglEwIgEAv+xCU/5BbnEAAAAASUVORK5CYII=","orcid":"","institution":"Universitat Autònoma de Barcelona (UAB)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Laia","middleName":"","lastName":"Aguirre","suffix":""},{"id":122669568,"identity":"d0c1a5d5-b326-4a18-bd4e-0804c1432d44","order_by":1,"name":"Yanli Li","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona (UAB)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Li","suffix":""},{"id":122669569,"identity":"a3932545-d011-46e0-99cf-bcbd82b8eaf4","order_by":2,"name":"Massimiliano Baratelli","email":"","orcid":"","institution":"HIPRA, Amer (Girona)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Massimiliano","middleName":"","lastName":"Baratelli","suffix":""},{"id":122669570,"identity":"bf4288bb-e15b-4cdf-80ab-47fb1c7aeedd","order_by":3,"name":"Gerard Martín-Valls","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona (UAB)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gerard","middleName":"","lastName":"Martín-Valls","suffix":""},{"id":122669571,"identity":"6b77b194-b02f-4c14-9106-c9aba36dc814","order_by":4,"name":"Martí Cortey","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona (UAB)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Martí","middleName":"","lastName":"Cortey","suffix":""},{"id":122669572,"identity":"42897e8d-10ae-42e0-bc1a-4d580001c6d0","order_by":5,"name":"Joel Miranda","email":"","orcid":"","institution":"HIPRA, Amer (Girona)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Joel","middleName":"","lastName":"Miranda","suffix":""},{"id":122669573,"identity":"be2c4ad8-9dc1-4140-a2d4-540ae5ab27e5","order_by":6,"name":"Marga Martín","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona (UAB)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marga","middleName":"","lastName":"Martín","suffix":""},{"id":122669574,"identity":"c096e9c7-0a97-43ca-af81-e63ea6cebcb8","order_by":7,"name":"Enric Mateu","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona (UAB)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Enric","middleName":"","lastName":"Mateu","suffix":""}],"badges":[],"createdAt":"2022-07-14 10:59:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1857671/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1857671/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24215428,"identity":"6c630504-ccc7-473b-8773-594a87c1212e","added_by":"auto","created_at":"2022-07-22 18:03:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProportion of PCR-positive pigs for PRRSV at different time-points after vaccination. \u003c/strong\u003eDifferences between groups were non-significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/a99f51ff3ed570befab47b77.png"},{"id":24215427,"identity":"e7f02f89-c978-417c-98a0-3683173e4df0","added_by":"auto","created_at":"2022-07-22 18:03:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolution of the S/P ratios in the different groups as determined by ELISA. \u003c/strong\u003eStatistically significant differences are indicated by different superscript letters.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/7a435fc9677b22a39a9ad831.png"},{"id":24215426,"identity":"79d65888-8cf5-4547-80e6-3c8cdb3a75bc","added_by":"auto","created_at":"2022-07-22 18:03:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of virus neutralisation titres in the different groups. \u003c/strong\u003eOnly statistically significant differences (p\u0026lt;0.05 are indicated, * p\u0026lt;0.05; ** p\u0026lt;0.01). Each dot represents one examined individual.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/7106cc6b959769dd6008a786.png"},{"id":24214689,"identity":"69ffa235-779f-4b5f-846d-f94167a096b5","added_by":"auto","created_at":"2022-07-22 17:58:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResults of the proliferation assays. \u003c/strong\u003eThe graphs depict the proportion of proliferating PBMC in cell cultures stimulated with the vaccine virus (21, 28 and 42 dpv, from left to right) at multiplicity of infection 0.1 once the spontaneous proliferation in mock-stimulated cultures was subtracted. Asterisks indicate the statistically significant differences (p\u0026lt;0.05). Each dot represents an individual.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/9921ad609c89989d525038a7.png"},{"id":24215429,"identity":"0919f5cd-54e0-48ab-b712-0a4b8da80ca4","added_by":"auto","created_at":"2022-07-22 18:03:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolution of the virus-specific IFN-γ responses as determined by ELISPOT. \u003c/strong\u003eThe graph depicts the frequencies of virus-specific IFN-γ secreting cells at 21, 28 and 42 dpv for the different groups. The bar indicates the median of each group. Differences between groups were non-significant. Each dot represents an individual.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/62f6d4f1f3b2c58daf744296.png"},{"id":24215564,"identity":"9298a9e5-848b-42fe-966a-e5939f7182c3","added_by":"auto","created_at":"2022-07-22 18:08:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":618008,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/01fcb1ad-0654-4ed3-99d2-c10c75483c0e.pdf"},{"id":24215563,"identity":"da761f30-c638-4a93-82ec-b4530f28feee","added_by":"auto","created_at":"2022-07-22 18:08:08","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material S1. Average Ct values for the vaccine-induced viremia a 7dpv. \u003c/strong\u003eThe graph depicts the average Ct-value of PCR-positive animals at 7 dpv (plus maximum, minimum, 25% and 75% quartiles). Different superscript letters indicated significant differences (p\u0026lt;0.05, Kruskal-Wallis test).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"S1.png","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/d0d93a217b84e4e78b3dfe2a.png"},{"id":24214684,"identity":"5240b71a-4f6f-4b3f-8a0a-88004df295a8","added_by":"auto","created_at":"2022-07-22 17:58:08","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material S2. Distribution of S/P ratios at the moment of the selection of seropositive animals. \u003c/strong\u003eThe graph depicts the distribution of S/P ratios for the group of animals sampled at 2 weeks of age for the selection of POS individuals.\u003c/p\u003e","description":"","filename":"S2.png","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/6791a65c1cf4178346458900.png"},{"id":24214686,"identity":"afc3e13f-a666-4381-8f0f-be1829e50f7e","added_by":"auto","created_at":"2022-07-22 17:58:08","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12277,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-1857671/v1/b917cc0c064e6f69efe627cc.docx"}],"financialInterests":"Competing interest reported. Massimiliano Baratelli and Joel Miranda are employees of Laboratorios Hipra.","formattedTitle":"Intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome 1 (PRRS1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells in the presence of maternally derived antibodies","fulltext":[{"header":"Background","content":"\u003cp\u003eThe porcine reproductive and respiratory syndrome (PRRS) is one of the main causes of economic loss in swine worldwide. Control of the infection is usually done by a combination of virus monitoring, biosecurity restrictions, herd management measures, and vaccination. Although vaccination most often targets sows, piglet vaccination is increasingly being considered to protect weaners in farms that have endemic problems in nurseries.\u003c/p\u003e \u003cp\u003eVaccination of piglets against PRRS virus (PRRSV) is usually performed during lactation or immediately after weaning. In endemically infected farms, most piglets present high levels of maternally derived antibodies (MDA) against PRRSV which may interfere with the development of an active immunity after vaccination [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The mechanisms of blocking by MDA are diverse (revised by Niewiesk [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]) and most often do not involve neutralization of the antigen but the interaction of MDA with B-cell receptors.\u003c/p\u003e \u003cp\u003eDifferent strategies can be used to avoid the eventual interference caused by maternal antibodies on vaccine immunisation. For instance, one common approach is to administer repeated doses of the vaccines (commonly two doses separated by a 3-4-week period) close to the age at which MDA are expected to wane to compensate for the eventual detrimental effect of the MDA on the first dose. Another option is to deliver the antigen to anatomical sites where the concentration of MDA is lower than the concentration in the muscle so there is less chance of interference with the vaccination. The intradermal route has several advantages such as the reduction of the antigen needed to induce an immune response [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and the potential of targeting a higher diversity of dendritic cells (reviewed by Combadiere and Liard [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eIn PRRS, the efficacy of vaccination by intradermal route has been proven to be similar to the intramuscular route [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] when pigs were not having specific MDA. However, much less is known about the efficacy of the intradermal route in the presence of MDA. The main objective of the present study was to compare the immune responses generated by a PRRSV live attenuated vaccine administered by either the intradermal or the intramuscular routes in piglets with high levels of specific MDA or in seronegative piglets.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e1. Clinical follow-up\u003c/h2\u003e\n\u003cp\u003eNo adverse reactions to vaccination were recorded. Two animals died during the study, one because of an internal haemorrhage (probably because of a fight) and the other one because of diarrhoea. Two animals were euthanised because of the development of bacterial meningitis. One additional animal was withdrawn from the study on arrival at the experimental facilities because of a lameness produced during the load and transport. No other incidents were observed in the rest of the animals during the 42-day follow-up.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2. Vaccine-induced viremia\u003c/h2\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the evolution of vaccine-induced viremia in the different groups. After vaccination animals were tested weekly to evaluate the presence of PRRS virus in blood. In all groups, vaccination produced a viremia at 7 days post-vaccination (dpv) (from 50\u0026ndash;91% of the vaccinated animals, depending on the group). After 21 dpv the proportion of viremic animals dropped significantly and after 35 dpv, only 1 or 2 animals/group were positive. No significant differences between groups were observed regarding the proportion of positive animals. Regarding Ct-values, significant differences were observed at 7 dpv between ID-POS and the IM-NEG with lower Ct values for the latter (Supplementary material S1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e\u003cem\u003e3. Development of antibodies\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe cohort of pigs tested in the seropositive herd (n\u0026thinsp;=\u0026thinsp;132) showed S/P values (ELISA) ranging between 0.44 and 2.24 (Supplementary material S2). The 28 selected animals had S/P values from 1.45 to 2.24. Those S/P values were in the quartile 75% of the distribution. At 4 weeks of age, when the vaccination was performed, animals in subgroup IM-POS had an S/P of 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 versus 1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 for ID-POS (non-significant differences). As expected, animals in group NEG were seronegative at the moment of vaccination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the evolution of average S/P values per group after vaccination. Vaccination produced a clear seroconversion in the seronegative animals (group NEG) that was noticeable by 14 dpv. Average S/P values increased until 35 dpv in the IM-NEG group, and until 42 dpv in the ID-NEG group. In the POS groups seroconversion was also observed, with increasing S/P ratios until day 28 dpv. It is worth noting that the ID-POS group presented the highest average S/P value at the end of the study, significantly different from that of the ID-NEG group. Differences between POS and NEG IM-vaccinated animals were non-significant.\u003c/p\u003e\n\u003cp\u003eSera obtained on the day of vaccination could neutralise the vaccine used in sows (titres of 2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42 log\u003csub\u003e2\u003c/sub\u003e and 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 log2 in IM and ID, respectively, non-significant) but they were devoid of neutralisation capacity against the vaccine administered to piglets. The development of neutralising antibody titres induced by the administration of the vaccine was firstly detected at 28 dpv. At 35 dpv, significant differences were noticed between the groups that were vaccinated in the presence of MDA and the seronegative ones (Fig.\u0026nbsp;3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thus, the animals of the seropositive origin presented higher titres compared to animals from the negative origin being IM or ID administration results equivalent.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4. Lymphoproliferation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePRRS-specific lymphocyte proliferation was clear by 21 dpv in some animals, which was more evident at 28 dpv and present in most animals at 35 dpv. Some differences in the proliferative responses were noticed at 35 dpv favouring the IM-NEG group (Kruskal-Wallis, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but they disappeared at 42 dpv (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e5. IFN-\u0026gamma; secreting cells\u003c/h2\u003e\n\u003cp\u003eELISPOT results were negative for all the animals at 0 dpv. Virus-specific responses were detected at 21 dpv in several animals from different groups and peaked at 42 dpv, with no significant differences between groups at any age (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e6. Cytokine production\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eIFN-\u0026alpha;.\u003c/em\u003e Levels of IFN-\u0026alpha; in serum at 7 dpv ranged between 300 and 700 pg/mL, with no significant differences between groups (IM-NEG: 435\u0026thinsp;\u0026plusmn;\u0026thinsp;83 pg/ml, ID-NEG: 420\u0026thinsp;\u0026plusmn;\u0026thinsp;79; ID-POS: 454\u0026thinsp;\u0026plusmn;\u0026thinsp;86; ID-POS: 481\u0026thinsp;\u0026plusmn;\u0026thinsp;86).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-10.\u003c/em\u003e At 7 dpv, levels of IL-10 were negative or very low (\u0026lt;\u0026thinsp;64 pg/ml) in all animals with no differences between groups. At 14 dpv values higher than 100 pg/ml of IL10 could be detected only in sera of NEG animals, 4 in the ID subgroup (164-1,849 pg/ml) and 2 in the IM subgroup (106\u0026ndash;553 pg/ml). Of these, at 21 dpv 2 animals still showed elevated IL10 levels in the ID-NEG group (249\u0026ndash;505 pg/ml) and one in the IM-NEG group (273 pg/ml). In culture supernatants, IL-10 concentration was very low and with no significant differences.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-12.\u003c/em\u003e At 7 dpv it was 10 times more likely to be IL-12 positive (9/28 vs. 1/23; p\u0026thinsp;=\u0026thinsp;0.033) for animals of the seronegative origin compared to the seropositive ones. At 14 dpv, all animals but three showed high levels of IL-12 in serum (195.1-3,821.6 pg/ml). Interestingly, of the 10 animals having the highest IL-12 levels at 14 dpv, 6 were the ones having elevated IL-10 at the same sampling time.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eInterference of MDA with vaccination is a major concern when young piglets are to be vaccinated. The mechanisms by which that interference occurs are not fully elucidated. However, there is strong evidence favouring the hypothesis that blocking by MDA may involve the inhibition of B-cell responses by the cross-linking between B cell receptor with the Fcγ-receptor IIB by a vaccine\u0026ndash;antibody complex, regardless of the neutralising capacity of the antibodies involved [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe intradermal administration of vaccines is an increasingly interesting alternative for vaccination, being less invasive and more respectful of animal welfare [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Interference with MDA could also be diminished by this vaccination route, as shown for the human poliovirus vaccine administered to children [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The skin has a rich diversity of antigen-presenting cells that are highly efficient for capturing and transporting antigens to the draining lymph nodes. Of special interest are Langerhans cells that are particularly able to induce cytotoxic T cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] but are less efficient in inducing responses from B cells. On the other hand, in humans, CD14\u003csup\u003e+\u003c/sup\u003e dermal dendritic cells are especially able to prime na\u0026iuml;ve B cells. This diversity of antigen-presenting cells in the skin can be advantageous for achieving effective immune responses after vaccination even when reduced amounts of antigen are available. Several studies proved that reduced doses of dermally-delivered antigens are effective for immunizing children with MDA against poliovirus [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the case of PRRSV, the intradermal administration of MLV PRRSV vaccines has proven to be as effective as the IM administration in na\u0026iuml;ve MDA-free pigs [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The vaccine tested in the present study showed that both routes were equally effective in developing immunity against PRRS in absence of MDA, which is in line with what showed by the above authors; moreover, the vaccine was demonstrated to be able to produce similar levels of immunity by means both routes even in presence of high MDA levels. It is worth mentioning here that, in PRRSV endemic farms, circulation of the virus often starts in the farrowing units because of the existence of vertical transmission from sows to newborns, or very soon after weaning since MDA wane between 4 and 5 weeks of age (reviewed by Pileri and Mateu [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]). Given the fast spread of the virus in na\u0026iuml;ve populations, vaccination of piglets must be administered at weaning or even before, implying that most vaccinated piglets will have MDA. Since the neutralising antibodies against PRRSV usually have a narrow breadth of neutralisation, the capability of MDA to neutralise a particular vaccine virus will depend on what vaccine was used in the sows and on their previous contact with other PRRSV strains.\u003c/p\u003e \u003cp\u003eThe most remarkable results were on animals of seropositive origin, which presented a stronger humoral response despite having high levels of MDA. This was clear for both S/P values (particularly for ID-POS) and virus neutralising antibodies (NA) titres.\u003c/p\u003e \u003cp\u003eIt is worth noting here that the scenario selected for the study was that where seropositive piglets did not have NA against the vaccine virus. Since NA in PRRS usually have a narrow range of neutralisation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], this scenario would account for most situations, including the vaccination of the offspring of sows vaccinated with a different vaccine, or simply, of seropositive unvaccinated sows. Although NA may have a role in blocking the replication of the MLV virus, it is known that vaccine blocking by the MDA is not necessarily related to the presence of NA [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis phenomenon of increased response to vaccination in individuals with sub-neutralising levels of antibodies has been proven in humans that were vaccinated with an experimental attenuated vaccine against the dengue virus [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In that case, the greater immune response in individuals with antibodies was attributed to increased infection of macrophages mediated by virus-antibody complexes. In the present case, Ct values of POS animals at 7 dpv were similar or lower to those of NEG animals. However, it is worth noting that there were more PCR-positive pigs at 7 dpv in the POS (IM and ID) compared to the NEG groups.\u003c/p\u003e \u003cp\u003eThe design could have certainly overlooked the case of animals with high homologous NA titres. Renson et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] showed that homologous neutralising MDA titres of about 1:10 may produce some interference with the development of immunity in piglets. The interference of MDA antibodies resulting from vaccination with different vaccines should be evaluated in the future.\u003c/p\u003e \u003cp\u003eRegarding the cell-mediated responses, the presence of MDA did not significantly interfere with the development of proliferative or IFN-γ responses. In other studies, it has been shown that vaccine blocking by MDA usually affects the development of humoral responses but not the development of cell-mediated immunity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present case, the development of vaccine-induced viremia was similar in all groups following a pattern described in other previous works [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. As shown before, some animals did not develop detectable viremia while others persisted as positive for several weeks. One possible element involved in this different behaviour could have been the IFN-α response. Renson et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] reported a potential correlation of this phenomenon with decreased response to the vaccine. This was not our case since all animals had comparable levels of this cytokine in serum.\u003c/p\u003e \u003cp\u003eInterestingly, the results showed that seronegative animals had a higher probability of having high levels of IL-12 in serum after vaccination than seropositive ones. This suggests a differential targeting of TLRs in animals with or without antibodies against the vaccine virus. Most of the animals with the highest IL-12 levels in serum were the ones with high IL-10 levels. A possible explanation could be in the homeostatic action of IL-10 o counterbalance excessive IL-12 production. It is known that IL-10 is a potent regulator of IL-12 transcription [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese results raise questions concerning the role of MDA in PRRS vaccination. The present results would indicate that in cases where the MDA were subneutralising (i.e., heterologous vaccines in sows and piglets), humoral immunity induced by MLV vaccination could even be enhanced. Further research should be done in this area to figure out what were the mechanisms leading to this difference including the possible enhancement of the vaccine virus replication and also whether vaccine-antibody complexes result in different stimulation of antigen-presenting cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, intradermal and intramuscular vaccinations have been proven as equivalent administration routes for the immunity parameters evaluated in this experimental study. The presence of non-neutralising antibodies at the time of vaccination resulted in enhanced humoral response without being detrimental to the development of cell-mediated responses.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1. Animals and experimental design\u003c/h2\u003e \u003cp\u003e The study was approved by the Committee for Ethics in Human and Animal Experimentation of the Universitat Aut\u0026ograve;noma de Barcelona (number CEEAH 5357) and by the Generalitat de Catalunya (FUE-2020-01836411).\u003c/p\u003e \u003cp\u003ePigs were recruited from two different herds; in the first, high levels of PRRSV-specific MDA were detected (group POS) whereas in the second no PRRSV antibodies were detected (group NEG). Group POS were DanBred x Pietrain crossbred animals. The farm was positive stable with vaccination according to the revised AASV scheme of classification as PRRSV was not detected at weaning [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. At two weeks of age, 132 piglets were randomly selected from the same farrowing batch and bled to determine the levels of MDA by ELISA (PRRS X3 Ab, Idexx). The absence of PRRSV circulation in the group was confirmed by RT-qPCR (VetMax PRRS EU\u0026amp;NA 2.0 RT-qPCR, ThermoFisher, Madrid, Spain). The 28 animals with the highest S/P ratios as determined by the test were selected for the experiment. Group NEG animals (n\u0026thinsp;=\u0026thinsp;28) were Duroc x Landrace pigs obtained from a historically PRRSV-free farm and its status was confirmed as well by ELISA. In both cases, animals were weaned at 3 weeks of age and transported to the experimental facilities located in the UAB. There, they were ear-tagged and randomly divided into two subgroups that were housed in physically separated boxes. One subgroup was assigned the intramuscular (IM) vaccination and the other was assigned the intradermal (ID) vaccination. Thus, the final design contained 4 subgroups: IM-POS, IM-NEG, ID-POS, and ID-NEG according to the serological status and the route of administration of the vaccine. Pigs were left to acclimate for a week before the administration of the vaccine.\u003c/p\u003e \u003cp\u003eAt 4 weeks of age, animals were bled and the PRRSV1 vaccine was administered (Unistrain\u0026reg; PRRS, Laboratorios Hipra). The IM vaccination was performed by the injection of 2 ml of the vaccine in the neck muscles; the ID vaccination was performed by delivering 0.2 ml of the vaccine in the neck using a needle-free device (Hipradermic\u0026reg; 3.0). Each dose of the vaccine contained 1x10\u003csup\u003e4.3\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e as titrated in MARC-145 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2. Sampling and sample processing\u003c/h2\u003e \u003cp\u003eAnimals were monitored for 42 days after vaccination. Blood samples were obtained weekly from all animals to determine the development of PRRSV-specific antibodies. Heparinized blood samples were additionally taken from 8 animals/group at 0-, 21-, 28-, and 42 dpv. The heparinized samples were used to obtain peripheral mononuclear blood cells (PBMC) by gradient density centrifugation using Histopaque-1077\u0026reg; (Sigma-Aldrich) and Sepmate\u0026trade; tubes (Stemcell Technologies, Saint-\u0026Eacute;gr\u0026egrave;ve, France). The resulting PBMC were frozen in Cryostor\u0026reg; CS10 (Merck) and stored in liquid nitrogen until further analysis. PBMC were recovered as described elsewhere [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and viability was checked by trypan blue staining (0.4%). Only samples with \u0026gt;\u0026thinsp;90% viability were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3. Serological analysis\u003c/h2\u003e \u003cp\u003eSerum samples were tested for the presence of PRRSV specific antibodies by HerdCheck\u0026reg; PRRS X3 Ab test Idexx ELISA.\u003c/p\u003e \u003cp\u003eViral neutralisation tests were performed in sera from 0-, 21-, 28-, 35-, and 42 dpv using the vaccine strain. For group POS animals, samples of 0 dpv were also tested against the vaccine strain used for the sows in the origin farm (Porcilis\u0026trade; PRRS; MSD Animal Health). The neutralisation tests were performed following the procedure described by Yoon \u003cem\u003eet al.\u003c/em\u003e (27) with minor modifications. Briefly, sera were inactivated at 56\u0026deg;C for 30 minutes and diluted from 1:2 to 1:256 in Minimum Essential Medium with non-essential amino acids, sodium pyruvate, 100 IU/mL penicillin and 100 \u0026micro;g/mL streptomycin. Equal volumes (100 \u0026micro;l:100 \u0026micro;l) of the diluted serum and the vaccine strain adjusted at 2,000 TCID\u003csub\u003e50\u003c/sub\u003e/mL were mixed and incubated overnight at 4\u0026deg;C. Then, 100 \u0026micro;L of the serum-virus mixture was transferred to MARC-145 monolayers in 96 well-plates. Negative (only medium) and virus controls (virus at working dilution and at 200, 20, and 2 TCID\u003csub\u003e50\u003c/sub\u003e/mL) were also included in the plate, as well as a negative and positive serum (post-vaccination serum of a sow) as controls. Plates were read after 6 days of incubation. The neutralisation titres were assessed by the development of the cytopathic effect and confirmed by immunofluorescence using the monoclonal antibody 1CH5 (Eurofins, Madrid). The neutralisation titre was the log\u003csub\u003e2\u003c/sub\u003e of the reciprocal of the highest dilution without cytopathic effect and without significant fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4. Quantification of PRRSV RNA\u003c/h2\u003e \u003cp\u003eQuantitative PCR for the quantification of PRRSV1 in serum was performed as previously reported [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5. Lymphoproliferation\u003c/h2\u003e \u003cp\u003eIsolated PBMC were stained with CellTrace\u0026trade; Violet (ThermoFisher) following the manufacturer's instructions. Briefly, 1x10\u003csup\u003e5\u003c/sup\u003e PBMC were dispensed on 96-well round-bottomed plates and the vaccine strain was added at a multiplicity of infection (MOI) of 0.1 (cultures were done in triplicate). In parallel, phytohemagglutinin-stimulated cultures (PHA, 10 \u0026micro;g/mL) or mock-stimulated cultures (RPMI medium) were used as positive and negative controls respectively. Plates were incubated for 5 days at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Then, cell cultures were recovered and centrifuged at 300 g for 10 min. The resulting pellet was resuspended in PBS and analysed in a Cytoflex flow cytometer (Beckman Coulter, CA, USA). Flow cytometer files were analysed using the FCS Express Flow Cytometry 6 (de Novo Software). For each sample, the PRRSV-specific proliferation was calculated as follows: percentage of cell proliferation in the PRRSV stimulated cultures - percentage of cell proliferation in the negative control. A relative proliferation index was also calculated as the proportion of cell proliferation in PRRSV-stimulated culture versus the proportion of cell proliferation in the mock-stimulated cultures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e6. IFN-γ ELISPOT\u003c/h2\u003e \u003cp\u003eIFN-γ ELISPOT was performed as described by Zuckerman \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] with minor modifications. Briefly, Costar 3590 plates (Corning) were coated with 50 \u0026micro;L of the monoclonal antibody P2G10 against porcine IFN-γ (ref. 559961; BD Pharmingen, NJ, USA) at 5 \u0026micro;g/mL diluted in carbonate-bicarbonate buffer (0.15M pH 9.5). After overnight incubation at 4\u0026deg;C, plates were washed five times with PBS and blocked for 1 hour with RPMI containing 10% foetal calf serum. Following the removal of the blocking solution, 50 \u0026micro;L/well of PBMC were dispensed at 1x10\u003csup\u003e5\u003c/sup\u003e PBMC/well (negative control and virus stimulated wells) and 5x10\u003csup\u003e4\u003c/sup\u003e PBMC/well (PHA-stimulated wells). PRRSV-stimulated wells were added to the vaccine strain at a MOI of 0.1. Mock-stimulated cultures (culture medium) and PHA-stimulated wells (10 \u0026micro;g/mL) were added as negative and positive controls, respectively. Plates were incubated overnight at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After washing, the biotinylated detection antibody P2C11 (ref. 559958; BD Pharmingen) was added at 0.5 \u0026micro;g/mL diluted in PBS with 0.4% bovine serum albumin. After a 1 hour incubation at 37\u0026deg;C, 50 \u0026micro;L of streptavidin-peroxidase was added at 0.5 \u0026micro;g/mL (ThermoFisher) and the reaction was developed by adding insoluble TMB (50 \u0026micro;L/well). Spots were counted in a Leica stereoscope after a 10-minute incubation in the dark. Samples were tested in triplicate. The frequencies of responding cells were calculated by subtracting the counts in the mock-stimulated wells from the counts obtained in virus-stimulated wells for each sample. Results were expressed as the number of responding cells per 1x10\u003csup\u003e6\u003c/sup\u003e PBMC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e7. Cytokine ELISA in culture supernatant and serum\u003c/h2\u003e \u003cp\u003eELISA for cytokines IL-10, IL-12 and IFN-α were performed in PBMC culture supernatants and/or serum. Cell culture supernatants were obtained from PBMC cultures produced in the same conditions as the ELISPOT. Supernatants were collected after overnight incubation of the PBMC and frozen at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis. Details of the samples analysed are shown in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eAntibody pairs and kits used are shown in supplementary materials (S3). Optimal work concentrations of each antibody were previously determined using the reference standards provided by each kit. Cytokine concentrations in the supernatants were estimated by calculating a regression formula obtained from the results produced by a serial dilution of the standard provided by the kit.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eEthics approval\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Committee for Ethics in Human and Animal Experimentation of the Universitat Aut\u0026ograve;noma de Barcelona (number CEEAH 5357) and by the Generalitat de Catalunya (FUE-2020-01836411).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eConsent for publication\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAvailability of data and materials\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCompeting interests\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMassimiliano Baratelli and Joel Miranda are employees of Laboratorios Hipra\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Laboratorios Hipra, S. A., 17170 Amer, Girona (Spain)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthors' contributions\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLaboratory work was performed by LA, YL, GMV and EM. Sample collection was done by GMV, MC and LA. Study design was performed by MM, EM, MB and JM. EM, GMV, MM and LA analysed results and wrote the manuscript. JM and MB reviewed the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRenson P, Fablet C, Andraud M, Normand V, Lebret A, Paboeuf F, et al. Maternally-derived neutralizing antibodies reduce vaccine efficacy against porcine reproductive and respiratory syndrome virus infection. Vaccine. 2019;37:4318\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFablet C, Renson P, Eono F, Mah\u0026eacute; S, Eveno E, Le Dimna M, et al. Maternally-derived antibodies (MDAs) impair piglets\u0026rsquo; humoral and cellular immune responses to vaccination against porcine reproductive and respiratory syndrome (PRRS). Vet Microbiol. Elsevier B.V.; 2016;192:175\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiewiesk S. Maternal antibodies: Clinical significance, mechanism of interference with immune responses, and possible vaccination strategies. Front Immunol. 2014;5:1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchnyder JL, Garcia Garrido HM, De Pijper CA, Daams JG, Stijnis C, Goorhuis A, et al. Comparison of equivalent fractional vaccine doses delivered by intradermal and intramuscular or subcutaneous routes: A systematic review. Travel Med Infect Dis. Elsevier Ltd; 2021;41:102007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCombadiere B, Liard C. Transcutaneous and intradermal vaccination. Hum Vaccin. 2011;7:811\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStadler J, Naderer L, Beffort L, Ritzmann M, Emrich D, Hermanns W, et al. Safety and immune responses after intradermal application of Porcilis PRRS in either the neck or the perianal region. PLoS One. 2018;13:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadapong A, Saeng-chuto K, Chaikhumwang P, Tantituvanont A, Saardrak K, Pedrazuela Sanz R, et al. Immune response and protective efficacy of intramuscular and intradermal vaccination with porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) modified live vaccine against highly pathogenic PRRSV-2 (HP-PRRSV-2) challenge, either alone or in co. Vet Microbiol. 2020;244.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartelli P, Gozio S, Ferrari L, Rosina S, De Angelis E, Quintavalla C, et al. Efficacy of a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs naturally exposed to a heterologous European (Italian cluster) field strain: Clinical protection and cell-mediated immunity. Vaccine. 2009;27:3788\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartelli P, Cordioli P, Alborali LG, Gozio S, De Angelis E, Ferrari L, et al. Protection and immune response in pigs intradermally vaccinated against porcine reproductive and respiratory syndrome (PRRS) and subsequently exposed to a heterologous European (Italian cluster) field strain. Vaccine. 2007;25:3400\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdwards KM. Maternal antibodies and infant immune responses to vaccines. Vaccine. Elsevier Ltd; 2015;33:6469\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Huey D, Oglesbee M, Niewiesk S. Insights into the regulatory mechanism controlling the inhibition of vaccine-induced seroconversion by maternal antibodies. Blood. 2011;117:6143\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTemple D, Escribano D, Jim\u0026eacute;nez M, Mainau E, Cer\u0026oacute;n JJ, Manteca X. Effect of the needle-free \u0026ldquo;intra dermal application of liquids\u0026rdquo; vaccination on the welfare of pregnant sows. Porc Heal Manag. Porcine Health Management; 2017;3:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSnider CJ, Zaman K, Estivariz CF, Yunus M, Weldon WC, Wannemuehler KA, et al. Immunogenicity of full and fractional dose of inactivated poliovirus vaccine for use in routine immunisation and outbreak response: an open-label, randomised controlled trial. Lancet. 2019;393:2624\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlechevsky E, Morita R, Liu M, Cao Y, Coquery S, Thompson-Snipes LA, et al. Functional Specializations of Human Epidermal Langerhans Cells and CD14 + Dermal Dendritic Cells. Immunity. 2008;29:497\u0026ndash;510.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMashunye TR, Ndwandwe DE, Dube KR, Shey M, Shelton M, Wiysonge CS. Fractional dose compared with standard dose inactivated poliovirus vaccine in children: a systematic review and meta-analysis. Lancet Infect Dis. Elsevier Ltd; 2021;21:1161\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrari L, Martelli P, Saleri R, De Angelis E, Cavalli V, Bresaola M, et al. Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs. Vet Immunol Immunopathol. Elsevier B.V.; 2013;151:193\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePileri E, Mateu E. Review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination. Vet Res. BioMed Central; 2016;47:1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Lobo FJ, D\u0026iacute;ez-Fuertes F, Simarro I, Castro JM, Prieto C. Porcine Reproductive and Respiratory Syndrome Virus isolates differ in their susceptibility to neutralization. Vaccine. 2011;29:6928\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillar L, Dayan GH, Arredondo-Garc\u0026iacute;a JL, Rivera DM, Cunha R, Deseda C, et al. Efficacy of a Tetravalent Dengue Vaccine in Children in Latin America. N Engl J Med. 2015;372:113\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHadinegoro SR, Arredondo-Garc\u0026iacute;a JL, Capeding MR, Deseda C, Chotpitayasunondh T, Dietze R, et al. Efficacy and Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease. N Engl J Med. 2015;373:1195\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGans HA, Yasukawa LL, Alderson A, Rinki M, DeHovitz R, Beeler J, et al. Humoral and cell-mediated immune responses to an early 2-dose measles vaccination regimen in the United States. J Infect Dis. 2004;190:83\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGans H, Yasukawa L, Rinki M, DeHovitz R, Forghani B, Beeler J, et al. Immune responses to measles and mumps vaccination of infants at 6, 9, and 12 months. J Infect Dis. 2001;184:817\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Lobo FJ, De Lome LC, D\u0026iacute;ez-Fuertes F, Segal\u0026eacute;s J, Garc\u0026iacute;a-Artiga C, Simarro I, et al. Safety of porcine reproductive and respiratory syndrome modified live virus (MLV) vaccine strains in a young pig infection model. 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A Modified Serum Neutralization Test for the Detection of Antibody to Porcine Reproductive and Respiratory Syndrome Virus in Swine Sera. J Vet Diagnostic Investig. 1994;6:289\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez E, Riera P, Sitj\u0026agrave; M, Fang Y, Oliveira S, Maldonado J. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green. Res Vet Sci. 2008;85:184\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuckermann FA, Husmann RJ, Schwartz R, Brandt J, Mateu De Antonio E, Martin S. Interleukin-12 enhances the virus-specific interferon gamma response of pigs to an inactivated pseudorabies virus vaccine. Vet Immunol Immunopathol. 1998;63:57\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"porcine-health-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phmj","sideBox":"Learn more about [Porcine Health Management](http://porcinehealthmanagement.biomedcentral.com/)","snPcode":"40813","submissionUrl":"https://submission.nature.com/new-submission/40813/3","title":"Porcine Health Management","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Porcine reproductive and respiratory syndrome virus, vaccine, Intramuscular, Intradermal, Maternally derived antibodies.","lastPublishedDoi":"10.21203/rs.3.rs-1857671/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1857671/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe purpose of this study was to compare the immune response generated by the intramuscular and the intradermal vaccination route against the porcine reproductive and respiratory syndrome virus (PRRSV). Piglets from a seronegative and a seropositive farm were selected (n\u0026thinsp;=\u0026thinsp;28 piglets per farm), and each group was divided into two groups and vaccinated at weaning with modified live vaccine Unistrain\u0026reg; PRRS (Laboratorios Hipra Amer, Spain) by the intramuscular or the intradermic route. For the following 6 weeks, animals were weekly bled to assess the humoral response by PRRSV-specific antibody ELISA and viral neutralisation test. At 0-, 3-, 4- and 6 weeks post-vaccination, peripheral mononuclear blood cells (PBMC) from eight animals per group were recovered to analyse cellular response by IFN-γ ELISPOT and lymphoproliferation. Serum IL-12 was also quantified by ELISA. Results showed no significant differences between treatments for any of the parameters studied. However, pigs from the seronegative origin had higher dispersion in S/P ratios by ELISA (Levene\u0026rsquo;s test for homogeneity of variances, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 3 weeks after vaccination, 6/27 (22.22%) animals from negative origin had not seroconverted. Also, it was 10 times more probable for them to have high levels of IL-12 a week after vaccination than for animals of seropositive origin. These results indicate that the intradermal route induces an immune response equivalent to the classical intramuscular route even in presence of maternal immunity, which in this study has proven to facilitate seroconversion after vaccination.\u003c/p\u003e","manuscriptTitle":"Intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome 1 (PRRS1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells in the presence of maternally derived antibodies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-22 17:58:06","doi":"10.21203/rs.3.rs-1857671/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-09-12T04:56:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-09-09T19:02:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-22T13:13:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"388d4651-d047-4840-8661-eb10e4531429","date":"2022-08-08T16:36:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-08-08T13:57:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-20T16:21:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-20T16:21:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Porcine Health Management","date":"2022-07-14T10:48:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"porcine-health-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phmj","sideBox":"Learn more about [Porcine Health Management](http://porcinehealthmanagement.biomedcentral.com/)","snPcode":"40813","submissionUrl":"https://submission.nature.com/new-submission/40813/3","title":"Porcine Health Management","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"45aa1fe1-2caf-46f1-846b-b09a7f0f1d36","owner":[],"postedDate":"July 22nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-11T09:44:29+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-22 17:58:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1857671","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1857671","identity":"rs-1857671","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}