An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity.

Oral Immunization With Plant-Based Vaccine Induces a Protective Response Against Infectious Bursal Disease

Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds causing important economic losses in the poultry industry worldwide. We have previously developed a plant-based vaccine candidate for infectious bursal disease (IBD) that is able to protect against infection with IBDV when administered through intramuscular (im) route. Given that oral vaccination is non-invasive and stimulates the immunity of the mucosal gastrointestinal surface, the initial site of contact and entry of IBDV, the aim of this work was to study if our immunogen was also able to elicit a protective immune response when orally administered. We demonstrated that 85% of the animals that received two oral doses of the vaccine formulation and all animals that were orally boosted after an im prime scheme developed virus neutralizing antibodies and were protected against IBDV infection, evidenced by the bursa/body weight (BB) ratio, absence of T-cell infiltration, and low viral load in bursa. Although mild to moderate bursal damage was observed in some of these animals, these lesions were not as severe as the ones observed in challenged control groups, which also presented signs of acute inflammation, bursal atrophy, T-cell infiltration, and absence of viral clearance. These results show that two immunizations with our recombinant immunogen are able to induce a specific and protective immune response in chicken against IBDV when orally administered in a prime/boost scheme or when the oral boost follows an im prime scheme. In conclusion, our oral plant-based vaccine candidate could represent a viable alternative to conventional vaccines and is of great interest to the poultry industry.

Effect of Paratuberculosis Vaccination on the in Vitro Release of Neutrophil Extracellular Traps in a Sheep Model

Background Vaccination of domestic ruminants against paratuberculosis has shown beneficial results in the control of the disease, even though protection is incomplete. This is partly due to the unawareness about the mechanisms taking part during protective immune response. In this sense, despite of the increased number of evidences that point out the participation of neutrophils in the homologous and heterologous protection, its key role is still unclear. Therefore, we evaluate the effect of paratuberculosis vaccination on the release of neutrophil extracellular traps (NETs), through its quantification and visualization after their in vitro infection with Mycobacterium avium subsp. paratuberculosis (Map) and non-related pathogens in a sheep model. Methods Ovine neutrophils were obtained from non-vaccinated (n = 5) and vaccinated sheep (n = 5) and infected in vitro with Map, Staphylococcus aureus (SA) and Escherichia coli (EC). After 4 hours, NETs release was quantified and visualized by fluorimetry and immunofluorescence microscopy, respectively. Results NETs components were confirmed by co-localization of extracellular DNA with neutrophil elastase and myeloperoxidase visualized by immunofluorescence. Infection of ovine neutrophils with Map (P > 0.05), SA (P < 0.001) and EC (P < 0.01) induced the formation of NETs before vaccination as well as after 15- and 30-days post-vaccination. However, neither vaccination nor time post-vaccination did modify the generation of NETs. Conclusions These results show that the release of NETs did not play a fundamental role in the early homologous and heterologous protective immune response after paratuberculosis vaccination, although, other antimicrobial mechanisms not evaluated in this study may participate in the protective immune response.

Preliminary evaluation of Brazilian green propolis as coadjuvant of a subunit vaccine against lethal leptospirosis

Vaccination would be preferred to leptospirosis control measures. However, current vaccines are heat killed whole-cell bacterins that generate serovar specific protection and several side effects. Modern molecular assays have revealed antigens that may replace traditional whole-cell vaccines. Among these, LigB protein is surface-exposed outer membrane protein of virulent leptospires and therefore potential target of a protective immune response. Some unsuccessful attempts at using these antigens as vaccines have been reported. However, we believe that immune modulation through alternative adjuvants and co-adjuvants may overcome previous setbacks. In this light, our study aimed to evaluate the protective immune response in hamsters vaccinated with 40 µg of rLigBNI using oil adjuvant (OA), with or without green propolis (GP) as co-adjuvant. Upon a challenge, all groups immunized with rLigBNI, coupled or not with GP, were highly immunogenic and revealed statistically significant (p<0.05) protection of hamsters from lethal leptospirosis. Additional studies are being carried out to assess the optimum dose, protection against heterologous challenge, and vaccine dynamics.

Radiation-Inactivated S. gallinarum Vaccine Provides a High Protective Immune Response by Activating Both Humoral and Cellular Immunity

Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Ebola Virus Glycoprotein Domains Associated with Protective Efficacy

Ebola virus (EBOV) is the cause of sporadic outbreaks of human hemorrhagic disease in Africa, and the best-characterized virus in the filovirus family. The West African epidemic accelerated the clinical development of vaccines and therapeutics, leading to licensure of vaccines and antibody-based therapeutics for human use in recent years. The most widely used vaccine is based on vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) (VSV-EBOV). Due to its favorable immune cell targeting, this vaccine has also been used as a base vector for the development of second generation VSV-based vaccines against Influenza, Nipah, and Zika viruses. However, in these situations, it may be beneficial if the immunogenicity against EBOV GP is minimized to induce a better protective immune response against the other foreign immunogen. Here, we analyzed if EBOV GP can be truncated to be less immunogenic, yet still able to drive replication of the vaccine vector. We found that the EBOV GP glycan cap and the mucin-like domain are both dispensable for VSV-EBOV replication. The glycan cap, however, appears critical for mediating a protective immune response against lethal EBOV challenge in mice.