Enhanced antigen-specific CD8 T cells contribute to early protection against FMDV through swine DC vaccination.

Foot-and-mouth disease virus (FMDV) remains a challenge for cloven-hooved animals. The currently licensed FMDV vaccines induce neutralizing antibody (NAb)-mediated protection but show defects in the early protection. Dendritic cell (DC) vaccines have shown great potency in inducing rapid T-cell immunity in humans and mice. Whether DC vaccination could enhance early protection against FMDV has not been elaborately explored in domestic pigs. In this study, we employed DC vaccination as an experimental approach to study the roles of cellular immunity in the early protection against FMDV in pigs. Autologous DCs were differentiated from the periphery blood mononuclear cells of each pig, pulsed with inactivated FMDV (iFMDV-DC) and treated with LPS, and then injected into the original pigs. The cellular immune responses and protective efficacy elicited by the iFMDV-DC were examined by multicolor flow cytometry and tested by FMDV challenge. The results showed that autologous iFMDV-DC immunization induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells (CTLs), high NAb titers, compared to the inactivated FMDV vaccine, and accelerated the development of memory CD4 and CD8 T cells, which was concomitantly associated with early protection against FMDV virulent strain in pigs. Such early protection was associated with the rapid proliferation of secondary T-cell response after challenge and significantly contributed by secondary CD8 effector memory T cells. These results demonstrated that rapid induction of cellular immunity through DC immunization is important for improving early protection against FMDV. Enhancing cytotoxic CD8+ T cells may facilitate the development of more effective FMDV vaccines.IMPORTANCEAlthough the currently licensed FMDV vaccines provide NAb-mediated protection, they have defects in early immune protection, especially in pigs. In this study, we demonstrated that autologous swine DC immunization augmented the cellular immune response and induced an early protective response against FMDV in pigs. This approach induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells, high NAb titers, and rapid development of memory CD4 and CD8 T cells. Importantly, the early protection conferred by this DC immunization is more associated with secondary CD8+ T response rather than NAbs. Our findings highlighted the importance of enhancing cytotoxic CD8+ T cells in early protection to FMDV in addition to Th1 response and identifying a strategy or adjuvant comparable to the DC vaccine might be a future direction for improving the current FMDV vaccines.

Within-Host Viral Growth and Immune Response Rates Predict Foot-and-Mouth Disease Virus Transmission Dynamics for African Buffalo.

AbstractInfectious disease dynamics operate across biological scales: pathogens replicate within hosts but transmit among populations. Functional changes in the pathogen-host interaction thus generate cascading effects across organizational scales. We investigated within-host dynamics and among-host transmission of three strains (SAT-1, -2, -3) of foot-and-mouth disease viruses (FMDVs) in their wildlife host, African buffalo. We combined data on viral dynamics and host immune responses with mathematical models to ask the following questions: How do viral and immune dynamics vary among strains? Which viral and immune parameters determine viral fitness within hosts? And how do within-host dynamics relate to virus transmission? Our data reveal contrasting within-host dynamics among viral strains, with SAT-2 eliciting more rapid and effective immune responses than SAT-1 and SAT-3. Within-host viral fitness was overwhelmingly determined by variation among hosts in immune response activation rates but not by variation among individual hosts in viral growth rate. Our analyses investigating across-scale linkages indicate that viral replication rate in the host correlates with transmission rates among buffalo and that adaptive immune activation rate determines the infectious period. These parameters define the virus's relative basic reproductive number (ℛ0), suggesting that viral invasion potential may be predictable from within-host dynamics.

A ferritin-based nanoparticle displaying a neutralizing epitope for foot-and-mouth disease virus (FMDV) confers partial protection in guinea pigs.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating disease affecting cloven-hoofed animals, that leads to significant economic losses in affected countries and regions. Currently, there is an evident inclination towards the utilization of nanoparticles as powerful platforms for innovative vaccine development. Therefore, this study developed a ferritin-based nanoparticle (FNP) vaccine that displays a neutralizing epitope of foot-and-mouth disease virus (FMDV) VP1 (aa 140-158) on the surface of FNP, and evaluated the immunogenicity and protective efficacy of these FNPs in mouse and guinea pig models to provide a strategy for developing potential FMD vaccines. RESULTS: This study expressed the recombinant proteins Hpf, HPF-NE and HPF-T34E via an E. coli expression system. The results showed that the recombinant proteins Hpf, Hpf-NE and Hpf-T34E could be effectively assembled into nanoparticles. Subsequently, we evaluated the immunogenicity of the Hpf, Hpf-NE and Hpf-T34E proteins in mice, as well as the immunogenicity and protectiveness of the Hpf-T34E protein in guinea pigs. The results of the mouse experiment showed that the immune efficacy in the Hpf-T34E group was greater than the Hpf-NE group. The results from guinea pigs immunized with Hpf-T34E showed that the immune efficacy was largely consistent with the immunogenicity of the FMD inactivated vaccine (IV) and could confer partial protection against FMDV challenge in guinea pigs. CONCLUSIONS: The Hpf-T34E nanoparticles stand out as a superior choice for a subunit vaccine candidate against FMD, offering effective protection in FMDV-infected model animals. FNP-based vaccines exhibit excellent safety and immunogenicity, thus representing a promising strategy for the continued development of highly efficient and safe FMD vaccines.

The Pathogenesis of Foot-and-Mouth Disease Virus Infection: How the Virus Escapes from Immune Recognition and Elimination.

Foot-and-mouth disease virus (FMDV) is a highly contagious and economically devastating pathogen that affects cloven-hoofed animals worldwide. FMDV infection causes vesicular lesions in the mouth, feet, and mammary glands, as well as severe systemic symptoms such as fever, salivation, and lameness. The pathogenesis of FMDV infection involves complex interactions between the virus and the host immune system, which determine the outcome of the disease. FMDV has evolved several strategies to evade immune recognition and elimination, such as antigenic variation, receptor switching, immune suppression, and subversion of innate and adaptive responses. This review paper summarizes the current knowledge on the pathogenesis of FMDV infection and the mechanisms of immune evasion employed by the virus. It also discusses the challenges and opportunities for developing effective vaccines and therapeutics against this important animal disease.

Monoclonal antibody based solid phase competition ELISA to detect FMDV serotype A specific antibodies.

In Foot-and-mouth disease (FMD) enzootic countries, periodic vaccination is the key tool in controlling the disease incidence. Active seromonitoring of the vaccinated population is critical to assess the impact of vaccination. Virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection. Assays like liquid phase blocking ELISA (LPBE) or solid phase competition ELISA (SPCE) are preferred as they do not require handling of live FMDV and are routinely used for seromonitoring or for vaccine potency testing; however, false positives are high in LPBE. Here we report, a monoclonal antibody (mAb) based SPCE as a potential alternate assay for antibody titration. From a panel of 12 mAbs against FMDV serotype A, two mAbs were chosen for the development of SPCE. Based on a set of 453 sera, it was demonstrated that mAb 2C4G11, mAb 6E8D11and polyclonal antibody (pAb) based SPCE had a relative sensitivity of 86.1, 86.1 and 80.3 %; and specificity of 99.6, 99.1 and 99.1 %, respectively. The correlation, repeatability, and level of agreement of the assays were high demonstrating the potential use of mAb in large scale surveillance studies and regular vaccine potency testing.

Effect of doubled dose administration of foot-and-mouth disease vaccine against heterologous virus infection in cattle.

Vaccination is a feasible approach for controlling foot-and-mouth disease (FMD). In FMD-free countries, vaccines are stored as a precautionary measure to control potential outbreaks. However, the challenge lies in pre-stocking optimal vaccines against the newly emerging strains. This study examined the potency of pre-stocked vaccines administered at elevated doses during emergencies. We vaccinated the cows with either a single or double trivalent vaccine dose containing two serotype O and one serotype A strains. Subsequently, vaccinated and unvaccinated cows were exposed to virulent strains of serotype O (O/JPN/2010; topotype Southeast Asia/Mya-98 lineage) or A (A/IRN/2016; topotype ASIA/G-VII lineage), which were genetically and antigenically distinct from the vaccine strains. Following challenge infections, all cows that received a single dose vaccination exhibited vesicular lesions with excreted viruses in the oral and nasal discharges. However, a substantial reduction was observed in the total clinical scores and virus titers in the sera and nasal discharges compared to those in the unvaccinated group. Cows receiving a doubled dose vaccination were completely protected from infection with O/JPN/2010 or demonstrated a significant decrease in viral shedding and clinical scores against A/IRN/2016. To note, vesicular lesions harbor significant amounts of viruses; thus, by mitigating their formation, viral transmission can be impeded, thereby slowing viral spread in the field. Furthermore, increasing the vaccine dose induced higher neutralizing antibody titers against heterologous strains. These findings suggest an alternative strategy for the effective management of future epidemics using pre-stocked vaccines.

Double synergic chitosan-coated poly (lactic-co-glycolic) acid nanospheres loaded with nucleic acids as an intranasally administered vaccine delivery system to control the infection of foot-and-mouth disease virus.

BACKGROUND & AIMS: The spread of foot-and-mouth disease virus (FMDV) through aerosol droplets among cloven-hoofed ungulates in close contact is a major obstacle for successful animal husbandry. Therefore, the development of suitable mucosal vaccines, especially nasal vaccines, to block the virus at the initial site of infection is crucial. PATIENTS AND METHODS: Here, we constructed eukaryotic expression plasmids containing the T and B-cell epitopes (pTB) of FMDV in tandem with the molecular mucosal adjuvant Fms-like tyrosine kinase receptor 3 ligand (Flt3 ligand, FL) (pTB-FL). Then, the constructed plasmid was electrostatically attached to mannose-modified chitosan-coated poly(lactic-co-glycolic) acid (PLGA) nanospheres (MCS-PLGA-NPs) to obtain an active nasal vaccine targeting the mannose-receptor on the surface of antigen-presenting cells (APCs). RESULTS: The MCS-PLGA-NPs loaded with pTB-FL not only induced a local mucosal immune response, but also induced a systemic immune response in mice. More importantly, the nasal vaccine afforded an 80% protection rate against a highly virulent FMDV strain (AF72) when it was subcutaneously injected into the soles of the feet of guinea pigs. CONCLUSIONS: The nasal vaccine prepared in this study can effectively induce a cross-protective immune response against the challenge with FMDV of same serotype in animals and is promising as a potential FMDV vaccine.

Extracellular vesicles derived from antigen-presenting cells pulsed with foot and mouth virus vaccine-antigens act as carriers of viral proteins and stimulate B cell response.

Foot and mouth disease (FMD) is a highly contagious infection caused by FMD-virus (FMDV) that affects livestock worldwide with significant economic impact. The main strategy for the control is vaccination with FMDV chemically inactivated with binary ethylenimine (FMDVi). In FMDV infection and vaccination, B cell response plays a major role by providing neutralizing/protective antibodies in animal models and natural hosts. Extracellular vesicles (EVs) and small EVs (sEVs) such as exosomes are important in cellular communication. EVs secreted by antigen-presenting cells (APC) like dendritic cells (DCs) participate in the activation of B and T cells through the presentation of native antigen membrane-associated to B cells or by transferring MHC-peptide complexes to T cells and even complete antigens from DCs. In this study, we demonstrate for the first time that APC activated with the FMDVi O1 Campos vaccine-antigens secrete EVs expressing viral proteins/peptides that could stimulate FMDV-specific immune response. The secretion of EVs-FMDVi is a time-dependent process and can only be isolated within the first 24 h post-activation. These vesicles express classical EVs markers (CD9, CD81, and CD63), along with immunoregulatory molecules (MHC-II and CD86). With an average size of 155 nm, they belong to the category of EVs. Studies conducted in vitro have demonstrated that EVs-FMDVi express antigens that can stimulate a specific B cell response against FMDV, including both marginal zone B cells (MZB) and follicular B cells (FoB). These vesicles can also indirectly or directly affect T cells, indicating that they express both B and T epitopes. Additionally, lymphocyte expansion induced by EVs-FMDVi is greater in splenocytes that have previously encountered viral antigens in vivo. The present study sheds light on the role of EVs derived from APC in regulating the adaptive immunity against FMDV. This novel insight contributes to our current understanding of the immune mechanisms triggered by APC during the antiviral immune response. Furthermore, these findings may have practical implications for the development of new vaccine platforms, providing a rational basis for the design of more effective vaccines against FMDV and other viral diseases.

Defining correlates of protection for mammalian livestock vaccines against high-priority viral diseases.

Enhancing livestock biosecurity is critical to safeguard the livelihoods of farmers, global and local economies, and food security. Vaccination is fundamental to the control and prevention of exotic and endemic high-priority infectious livestock diseases. Successful implementation of vaccination in a biosecurity plan is underpinned by a strong understanding of correlates of protection-those elements of the immune response that can reliably predict the level of protection from viral challenge. While correlates of protection have been successfully characterized for many human viral vaccines, for many high-priority livestock viral diseases, including African swine fever and foot and mouth disease, they remain largely uncharacterized. Current literature provides insights into potential correlates of protection that should be assessed during vaccine development for these high-priority mammalian livestock viral diseases. Establishment of correlates of protection for biosecurity purposes enables immune surveillance, rationale for vaccine development, and successful implementation of livestock vaccines as part of a biosecurity strategy.

Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice.

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

A recombinant multi-epitope trivalent vaccine for foot-and-mouth disease virus serotype O in pigs.

In order to develop a safe and effective broad-spectrum vaccine for foot-and-mouth disease (FMDV), here, we developed a recombinant FMD multiple-epitope trivalent vaccine based on three distinct topotypes of FMDV. Potency of the vaccine was evaluated by immune efficacy in pigs. The results showed that the vaccine with no less than 25 µg of antigen elicited FMDV serotype O specific antibodies and neutralization antibodies by primary-booster regime, and offered immune protection to pigs. More importantly, the vaccine elicited not only the same level of neutralization antibodies against the three distinct topotypes of FMDV, but also provided complete protection in pigs from the three corresponding virus challenge. None of the fully protected pigs were able to generate anti-3ABC antibodies throughout the experiment, which implied the vaccine can offer sterilizing immunity. The vaccine elicited lasting-long high-level antibodies and effectively protected pigs from virulent challenge within six months of immunization. Therefore, we consider that this vaccine may be used in the future for the prevention and control of FMD.

Virus-like particle-based multipathogen vaccine of FMD and SVA elicits balanced and broad protective efficacy in mice and pigs.

Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.

Oral immunization against foot-and-mouth disease virus using recombinant Saccharomyces cerevisiae with the improved expression of the codon-optimized VP1 fusion protein.

VP1, a major immunogenic protein of foot-and-mouth disease virus (FMDV), facilitates viral attachment and entry into host cells. VP1 possesses critical epitope sequences responsible for inducing neutralizing antibodies but its expression using Saccharomyces cerevisiae has been hampered despite evidence that the presence of VP1 does not negatively impact the yeast's biology. In this study, we fused proteins to enhance VP1 expression using S. cerevisiae. Among short P1 chimeras containing VP1 including VP3-VP1 and VP2-VP1, VP3-VP1 fusion proteins showed higher expression levels than VP2-VP1. We subsequently designed new fusion proteins, of which 20 amino acids of N-terminal VP3 fused with VP1-Co1 (referred to 20aaVP3-VP1-Co1) showed the highest expression level. Lowering the culture temperature from 30 °C to 20 °C further enhanced fusion protein production. The highest expression level of 20aaVP3-VP1-Co1 was estimated to be 7.7 mg/L, which is comparable to other heterologous proteins produced using our S. cerevisiae expression system. Oral administration of the cell expressing 20aaVP3-VP1-Co1 induced VP1-specific IgG and IgA responses in mice. The S. cerevisiae-expressed 20aaVP3-VP1-Co1 fusion protein induced a significant immune response to the FMDV structural epitope protein, which opens the possibility of an oral FMDV vaccine.

Compared to Aluminum Hydroxide Adjuvant, Montanide ISA 206 VG Induces a Higher and More Durable Neutralizing Antibody Response against FMDV in Goats.

Foot-and-mouth disease (FMD) has a high prevalence in cloven-hoofed animals. It is also highly contagious and remains a serious threat to livestock worldwide. Despite the widespread vaccination program in Iran, outbreaks of FMD continue to occur. Vaccination is one of the most effective methods of preventing FMD. The vaccines used in Iran are of the inactivated type and contain several serotypes. Since inactivated vaccines without adjuvants do not induce a high and durable antibody response, it is necessary to use adjuvants. Montanide ISA 206 VG is a mineral oil-based adjuvant that produces a water-in-oil-in-water (w:o:w) emulsion in vaccine preparations. However, a large number of manufacturers in Iran and around the world still use alum adjuvant (with or without saponin) to produce the FMD vaccine. This study used Montanide ISA 206 and alum adjuvants to administer the O2010 serotype of the FMD virus to goats. A total of six goats were divided randomly into three groups. Vaccines were administered subcutaneously twice, at a one-month interval. Blood sampling was done at different times, and the micro-neutralization method was used to measure the neutralizing antibody titer in each serum. Seven days after the second vaccination, the alum group's antibody titer was higher but not statistically significant. However, from the 28th day after the second injection until the end of the study, the Montanide ISA 206 group's antibody titer was significantly higher than that of the alum group. Six months after the second injection, the antibody titer in the ISA 206 group remained at the peak level, while in the alum group, it decreased and reached the minimum protective level. Nine months after the second injection, the antibody titer remained at its peak level in the ISA 206 group, whereas it dropped significantly in the alum group. Based on the findings, ISA 206 VG is capable of generating long-term humoral immunity in goats against the FMD serotype O2010 and could replace aluminum hydroxide adjuvants in FMD vaccine preparations.

A pro-inflammatory effect of foot and mouth disease virus on immune and non immune guinea pigs

A estirpe O 1campos do vírus da febre aftosa (VFA) usada como agente indutor do teste de pleurisia foi capaz de desencadear um efeito pró-inflamatório em cobaias normais e imunes. A atividade pró-inflamatória do VFA, detectada em dois intervalos de pleurisia (24 e 48 horas) foi demonstrada, somente por quimiotaxia de leucócitos mononucleares (MN), no primeiro intervalo e por efeito edematogênico, migraçäo de MN e polimorfonucleares (PMN), no último intervalo de reaçäo. Os perfis de reaçäo inflamatória induzida pelo VFA em cobaias imunes (imunizadas com vacinas oleosas anti-VFAO 1 campos), aos 7 e aos 30 dias pós-vacinaçäo (PV) apresentaram intensidades mais baixas do que as observadas em cobaias normais, embora nas cobaias com 7 dias de vacinaçäo a quimiotaxia de leucócitos totais e de PMN tenha sido similar àquela encontrada nos animais normais, no intervalo de 48 horas de reaçäo. Ademais, nas cobaias com 30 dias PV, o VFA induziu um aumento significante no volume de exsudato e na infiltraçäo de MN, no intervalo de 24 horas, sendo que os valores de todos os parâmetros do exsudato inflamatório caíram a níveis normais, no segundo intervalo de reaçäo. Nas cobaias imunes foi observada uma associaçäo negativa entre o aumento no título de anticorpos soro-neutralizantes, de 7 para 30 dias PV e as intensidades dos parâmetros inflamatórios pleurais. O teste de pleurisia revelou-se um procedimento adequado para avaliar a atividade pró-inflamatória do VFA

Efficiency test for foot and mouth disease vaccine. II. Relationship between the C index in Guinea pigs and the mouse protection index

O exame da eficiência de seis vacinas antiaftosa, realizado com os testes "Indice Proteçäo Camundongos" e "Indice C" utilizando 15 ou mais cobaias por diluiçäo de vírus na titulaçäo, demonstrou a presença de boa correlaçäo e alta significância entre os resultados das duas provas. O valor do Indice Proteçäo Camundongos foi aproximadamente o dobro do valor do Indice C. Assim, o Indice Proteçäo Camundongos pode ser recomendado como teste de eficiência de vacians antiaftosa, principalmente quando se necessita de um grande número de provas, devido a seu baixo custo e facilidade de execuçäo, por usar exclusivamente camundongos

Comportamiento de una vacuna antiaftosa inactivada oleosa para cerdos, producida en Venezuela / Behaviour of inactivated foot and mouth oily vaccine for pigs

Se realizó el estudio de una vacuna antiaftosa inactivada oleosa para cerdos (AFTO-VAC), a fin de conocer su comportamiento. Establecida su esterilidad e inocuidad se comprobó, mediante la prueba de fijación de complemento, que la vacuna fue elaborada con los subtipos O1, A24 y A32, y que los títulos serológicos eran de 1/6, 1/2 y 1/2 para cada uno, respectivamente. Para estimar la potencia de la vacuna frente a cada subtipo, se utilizó la prueba de Protección a la Generalización Podal (P.G.P.) en cerdos, considerándose satisfactorio cuando la protección alcanzó al menos el 60 por ciento de los animales confrontados. La confrontación se realizó con 100 dosis generalizantes 50 por ciento en cerdo, de virus patógenos de fiebre aftosa subtipos O1, A24, A32. Observándose 100 por ciento, 58 por ciento y 60 por ciento de protección para cada subtipo, respectivamente. Los niveles de anticuerpos circulantes en los cerdos al momento de la confrontación fueron determinados mediante la prueba de Protección en Ratón Lactante y expresados en valores de Indices de Seroprotección (I.S.P.). Estos resultados serológicos mostraron que bajos niveles de I.S.P. no necesariamente indican ausencia de protección