Inactivated Whole-virus Research Articles

The type of adjuvant in whole inactivated influenza a virus vaccines impacts vaccine-associated enhanced respiratory disease

Influenza A virus (IAV) causes a disease burden in the swine industry in the US and is a challenge to prevent due to substantial genetic and antigenic diversity of IAV that circulate in pig populations. Whole inactivated virus (WIV) vaccines formulated with oil-in-water (OW) adjuvant are commonly used in swine. However, WIV-OW are associated with vaccine-associated enhanced respiratory disease (VAERD) when the hemagglutinin and neuraminidase of the vaccine strain are mismatched with the challenge virus. Here, we assessed if different types of adjuvant in WIV vaccine formulations impacted VAERD outcome. WIV vaccines with a swine δ1-H1N2 were formulated with different commercial adjuvants: OW1, OW2, nano-emulsion squalene-based (NE) and gel polymer (GP). Pigs were vaccinated twice by the intramuscular route, 3 weeks apart, then challenged with an H1N1pdm09 three weeks post-boost and necropsied at 5 days post infection. All WIV vaccines elicited antibodies detected using the hemagglutination inhibition (HI) assay against the homologous vaccine virus, but not against the heterologous challenge virus; in contrast, all vaccinated groups had cross-reactive IgG antibody and IFN-γ responses against H1N1pdm09, with a higher magnitude observed in OW groups. Both OW groups demonstrated robust homologous HI titers and cross-reactivity against heterologous H1 viruses in the same genetic lineage. However, both OW groups had severe immunopathology consistent with VAERD after challenge when compared to NE, GP, and non-vaccinated challenge controls. None of the WIV formulations protected pigs from heterologous virus replication in the lungs or nasal cavity. Thus, although the type of adjuvant in the WIV formulation played a significant role in the magnitude of immune response to homologous and antigenically similar H1, none tested here increased the breadth of protection against the antigenically-distinct challenge virus, and some impacted immunopathology after challenge.

Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens

Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines.

Gene cloning, expression and immune adjuvant properties of the recombinant fusion peptide Tα1-BLP on avian influenza inactivate virus vaccine

Thymosin α1 (Tα1) and bursin-like peptide (BLP) are both immunopotentiators. In order to investigate adjuvant of thymosin α1-bursin-like peptide (Tα1-BLP), we cloned the gene of Tα1-BLP and provided evidence that the gene of Tα1-BLP in a recombinant prokaryotic expression plasmid was successfully expressed in E. coli BL21. To evaluate the immune adjuvant properties of Tα1-BLP, chickens were immunized with Tα1-BLP combined with H9N2 avian influenza whole-inactivated virus (WIV). The titers of HI antibody, antigen-specific antibodies, AIV-neutralizing antibodies, levels of Th1-type cytokines (IFN-γ) and Th2-type cytokines (IL-4) and lymphocyte proliferation responses were determined. What's more, the viral loads and pathologic changes of lung tissue were observed by virus challenge experiment and HE staining to evaluate the immune protection of chickens. We found that Tα1-BLP enhanced HI antibody and antigen-specific IgG antibodies titers, increased the level of AIV-neutralizing antibodies, induced the secretion of Th1- and Th2-type cytokines, and promoted the proliferation of T and B lymphocyte, Furthermore, virus challenge experiment and HE staining confirmed that Tα1-BLP contributed to inhibition replication of the virus from chicken lungs and protected the lungs from damage. Altogether, this study suggested that Tα1-BLP is a novel adjuvant suitable for H9N2 avian influenza vaccine.

Development and Utilization of VHH Antibodies Derived from Camelus Dromedarius Against Foot-and-Mouth Disease Virus

ABSTRACTFoot-and-mouth disease (FMD) is an acute, highly contagious, and economically devastating viral disease of domestic and wildlife species. For effective implementation of FMD control program, there is an imperative need for developing a rapid, sensitive, and specific diagnostics which help in the identification of serotypes involved in the outbreaks. The humoral immune response of the Camelidae is unique since in these animals 75% of circulating antibodies are constituted by heavy-chain antibodies and 25% are conventional immunoglobulin with two identical heavy chains. In the present study, we developed and characterized FMD virus-specific single-domain heavy-chain antibodies (VHHs) against inactivated whole-virus antigens of FMDV serotypes O (INDR2/1975), A (IND40/2000), and Asia 1 (IND63/1972) vaccine strains. After six rounds of panning and enrichment, these VHHs were stably expressed in Escherichia coli cells. The VHHs directed against outer capsid proteins of FMD virus were successfully utilized as the capture antibody in liquid-phase blocking ELISA (LPBE) thus replacing rabbit coating antibodies. Our study demonstrated the utility of FMD virus-specific VHHs as potential candidates in FMD research and diagnostic application.

Sex bias in mouse humoral immune response to influenza vaccine depends on the vaccine type

The study explored influence of biological sex on development of humoral immune response to seasonal trivalent whole inactivated virus (WIV) and split virus (SV) influenza vaccines in outbred Swiss mouse model. To this end, mice of both sexes were immunized with WIV (WIV mice) and SV vaccines (SV mice) and examined for specific antibody response. Irrespective of sex, total IgG and neutralizing antibody responses to distinct virus strains were weaker in SV than in WIV mice. In WIV mice of both sexes, irrespective of strain specificity, IgG isotype response was dominated by IgG2a antibodies, while in SV mice nearly equal representation of IgG2a and IgG1 antibodies was found. The analyses of sex differences showed higher titers of H1N1-specific and both H1N1- and H3N2-specific total IgG and neutralizing antibodies in female WIV and SV mice, respectively. Additionally, sexual dimorphism in IgG subclass profile depended on vaccine type. Specifically, compared with males, in females WIV shifted IgG2a/IgG1 antibody ratio towards IgG2a isotype on the account of weaker IgG1 response, whereas in SV mice, irrespective of virus strain, IgG2a and IgG1 isotypes were equally represented in both sexes. These findings indicate the vaccine type-dependent sex bias in antibody response to inactivated influenza vaccines.

Zika Virus Vaccine Development.

The emergence of Zika virus in Brazil and its association with microcephaly and Guillain-Barré syndrome led to accelerated vaccine development efforts. Based on prior flavivirus vaccine development programs, knowledge of flavivirus particle structure, definition of E dimers as the key antigenic target, and deep understanding of neutralizing mechanisms, multiple vaccine strategies have advanced to the stage of clinical evaluation with unprecedented speed. These include nucleic acid (DNA and messenger RNA), whole-inactivated virus, live-attenuated or chimeric virus, and protein or viruslike particle vaccines. Within a year from the declaration by the World Health Organization of Zika virus as a Public Health Emergency of International Concern, multiple vaccine candidates entered clinical trials, now totaling 7 products with an additional 40-plus candidate vaccines in preclinical development. The rapid progress in vaccine development demonstrates the capacity of governments, public health organizations, and the scientific community to respond to pandemic threats when sufficient prior knowledge exists, emergency funding is made available, and interagency cooperation is achieved and serves as a paradigm for preparing for future emerging infectious diseases.

Research progress in human infection with avian influenza H7N9 virus.

Since the identification of the novel reassortant avian influenza A (H7N9) virus in China in 2013, until Jun 30, 2017, the virus has caused five epidemic waves leading to a total of 1,552 human infections, with a fatality rate of about 40%. In the spring of 2017, highly pathogenic avian influenza (HPAI) H7N9 virus emerged and has caused 25 human infections. The HPAI H7N9 virus has some biological differences from the LPAI one, such as its multiple basic amino acid residues on HA leading to its independence on trypsin for replication. The pathogenicity of the HPAI H7N9 virus to experimental animals or humans is still unclear. A(H7N9) vaccine development for pandemic preparedness is ongoing, including the reassortment (H7N9/PR8) reverse genetic based vaccine, the virus like particle (VLP) vaccine, the intranasal live attenuated influenza vaccine (LAIV), the non-adjuvant Vero cell culture-derived inactivated whole-virus vaccine, the MDCK culture-derived vaccine, the H7 DNA vaccine and the recombinant replicative H7N9 virus (H7N9-53TM) vaccine. Five neuramidinase resistant sites of A(H7N9) virus isolated from patients have been reported. Some alternative drugs have been studied, such as DAS181 (Fludase), ribavirin, troglitazone and minocycline. Persistent surveillance and enhanced global control are essential to fight against human infections with A(H7N9) virus.

Single immunization with MF59-adjuvanted inactivated whole-virion H7N9 influenza vaccine provides early protection against H7N9 virus challenge in mice

H7N9 influenza infection in humans would result in severe respiratory illness. Vaccination is the best way to prevent influenza virus. In this paper, we investigated the effect of early protection provided by inactivated whole-virion H7N9 influenza vaccine in a mouse model.Mice were immunized intramuscularly once with different doses of inactivated whole-virion H7N9 influenza vaccine alone or in combination with MF59 adjuvant. Specific IgM and IgG antibody titers in sera of mice were detected by ELISA 3, 5 and 7days after immunization. To evaluate the early protection provided by the vaccine, mice were challenged with lethal dose (40LD50) of homologous virus 3, 5 and 7 days after immunization respectively. The survival rate and body weight change of mice during 21 days after challenge and the residual lung virus titer on 3rd day after challenge were determined. The results demonstrated that mice could obtain effective protection 3 days after immunization with the vaccine at a high dose, and 5–7 days after immunization even at a low dose. Thus early immune responses induced by inactivated whole-virion H7N9 vaccine could provide effective protection.

Distinctive Responses in an In Vitro Human Dendritic Cell-Based System upon Stimulation with Different Influenza Vaccine Formulations.

Vaccine development relies on testing vaccine candidates in animal models. However, results from animals cannot always be translated to humans. Alternative ways to screen vaccine candidates before clinical trials are therefore desirable. Dendritic cells (DCs) are the main orchestrators of the immune system and the link between innate and adaptive responses. Their activation by vaccines is an essential step in vaccine-induced immune responses. We have systematically evaluated the suitability of two different human DC-based systems, namely the DC-cell line MUTZ-3 and primary monocyte-derived DCs (Mo-DCs) to screen immunopotentiating properties of vaccine candidates. Two different influenza vaccine formulations, whole inactivated virus (WIV) and subunit (SU), were used as model antigens as they represent a high immunogenic and low immunogenic vaccine, respectively. MUTZ-3 cells were restricted in their ability to respond to different stimuli. In contrast, Mo-DCs readily responded to WIV and SU in a vaccine-specific way. WIV stimulation elicited a more vigorous induction of activation markers, immune response-related genes and secretion of cytokines involved in antiviral responses than the SU vaccine. Furthermore, Mo-DCs differentiated from freshly isolated and freeze/thawed peripheral blood mononuclear cells (PBMCs) showed a similar capacity to respond to different vaccines. Taken together, we identified human PBMC-derived Mo-DCs as a suitable platform to evaluate vaccine-induced immune responses. Importantly, we show that fresh and frozen PBMCs can be used indistinctly, which strongly facilitates the routine use of this system. In vitro vaccine pre-screening using human Mo-DCs is thus a promising approach for evaluating the immunopotentiating capacities of new vaccine formulations that have not yet been tested in humans.

Factors affecting induction of peripheral IFN-γ recall response to influenza A virus vaccination in pigs

While T cell contribution to IAV immunity is appreciated, data comparing methods to evaluate IFN-γ production by IAV-specific T cells elicited following vaccination is limited. To understand the differential immunogenicity between live-attenuated influenza virus (LAIV) and whole-inactivated virus (WIV) vaccines in relation to induction of peripheral T cell responses, ELISpot and ELISA were used to assess IFN-γ production by peripheral lymphocytes following antigen restimulation. Following restimulation, peripheral blood lymphocytes from WIV-vaccinated pigs had a greater quantity of IFN-γ secreting cells (SC) and IFN-γ secreted compared to LAIV vaccinated and non-vaccinated (NV) pigs. Pig age at time of WIV vaccination significantly impacted peripheral IAV-specific IFN-γ recall response, as did the inclusion of adjuvant in the WIV vaccine. Collectively, these data indicate that peripheral IAV-specific IFN-γ recall responses are not predictive of LAIV vaccination status, thus, are unlikely to be a useful surrogate for evaluating LAIV immunogenicity and predicting cross-protection. However, these data suggest that the evaluation of peripheral IFN-γ recall responses may be useful for identifying factors, such as animal age or vaccine formulation, that may impact parenterally-delivered WIV vaccine immunogenicity. Overall, results did not differ based upon the assay used to evaluate IFN-γ recall responses. Therefore, either ELISpot or ELISA could serve as a measure for evaluating IAV-specific IFN-γ cell-mediated immune responses in swine.

A virus-like particle vaccination strategy expands its tolerance to H3N2 antigenic drift by enhancing neutralizing antibodies against hemagglutinin stalk

Seasonal influenza viruses impact public health annually due to their continual evolution. However, the current inactivated seasonal vaccines provide poor protection against antigenically drifted viruses and require periodical reformulation through hit-and-miss predictions about which strains will circulate during the next season. To reduce the impact caused by vaccine mismatch, we investigated the drift-tolerance of virus-like particles (VLP) as an improved vaccine candidate. The cross-protective humoral immunity elicited by the H3N2-VLP vaccine constructed for the 2011–2012 season was examined against viruses isolated from 2010 to 2015 in Taiwan evolving chronologically through clades 1, 4, 5, 3B and 3C, as well as viruses that were circulating globally in 2005, 2007 and 2009. Mouse immunization results demonstrated that H3N2-VLP vaccine elicited superior immunological breadth in comparison with the cognate conventional whole-inactivated virus (WIV) vaccine. Titers of neutralizing antibodies against heterologous strains representing each epidemic period in the VLP group were significantly higher than in the WIV group, indicating the antibody repertoire induced by the H3N2-VLPs was insensitive to viral antigenic drift over a span of at least 10 years. Noticeably, H3N2-VLP elicited higher levels of anti-stalk antibodies than H3N2-WIV, which offset the ineffectiveness caused by antigenic drift. This advantageous effect was attributed to the uncleaved precursor of their HA proteins. These results suggest a mechanism through which VLP-induced humoral immunity may better tolerate the evolutionary dynamics of influenza viruses and point to the possible use of a VLP vaccine as a method by which the requirement for annual updates of seasonal influenza vaccines may be diminished.

A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1:20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.

Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation.

Coxsackievirus A16 (CA16) is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) in the Asia-pacific region. Although CA16 infections are generally mild, severe neurological manifestations or even death has been reported. Studies on CA16 pathogenesis and vaccine development are severely hampered because the small animal models that are currently available show major limitations. In this study, gerbils (Meriones unguiculatus) were investigated for their suitability as an animal model to study CA16 pathogenesis and vaccine development. Our results showed that gerbils up to the age of 21 days were fully susceptible to CA16 and all died within five days post-infection. CA16 showed a tropism towards the skeletal muscle, spinal cord and brainstem of gerbils, and severe lesions, including necrosis, were observed. In addition, an inactivated CA16 whole-virus vaccine administrated to gerbils was able to provide full protection to the gerbils against lethal doses of CA16 strains. These results demonstrate that gerbils are a suitable animal model to study CA16 infection and vaccine development.

The Current Status of the Disease Caused by Enterovirus 71 Infections: Epidemiology, Pathogenesis, Molecular Epidemiology, and Vaccine Development.

Enterovirus 71 (EV71) infections have a major public health impact in the Asia-Pacific region. We reviewed the epidemiology, pathogenesis, and molecular epidemiology of EV71 infection as well as EV71 vaccine development. Previous studies were found using the search terms “enterovirus 71” and “epidemiology” or “pathogenesis” or “molecular epidemiology” or “vaccine” in Medline and PubMed. Articles that were not published in the English language, manuscripts without an abstract, and opinion articles were excluded from the review. The reported epidemiology of cases caused by EV71 infection varied from country to country; seasonal variations in incidence were observed. Most cases of EV71 infection that resulted in hospitalization for complications occurred in children less than five years old. The brainstem was the most likely major target of EV71 infection. The emergence of the EV71 epidemic in the Asia-Pacific region has been associated with the circulation of different genetic lineages (genotypes B3, B4, C1, C2, and C4) that appear to be undergoing rapid evolutionary changes. The relationship between the gene structure of the EV71 virus and the factors that ensure its survival, circulation, and evasion of immunity is still unknown. EV71 infection has emerged as an important global public health problem. Vaccine development, including the development of inactivated whole-virus live attenuated, subviral particles, and DNA vaccines, has been progressing.

Inactivated coxsackievirus A10 experimental vaccines protect mice against lethal viral challenge

Coxsackievirus A10 (CVA10) has become one of the major causative agents of hand, foot and mouth disease (HFMD). It is now recognized that CVA10 should be targeted for vaccine development. We report here that β-propiolactone inactivated whole-virus based CVA10 vaccines can elicit protective immunity in mice. We prepared two inactivated CVA10 experimental vaccines derived from the prototype strain CVA10/Kowalik and from a clinical isolate CVA10/S0148b, respectively. Immunization with the experimental vaccines elicited CVA10-specific serum antibodies in mice. The antisera from vaccinated mice could potently neutralize in vitro infection with either homologous or heterologous CVA10 strains. Importantly, passive transfer of the anti-CVA10 sera protected recipient mice against CVA10/Kowalik or CVA10/S0148b infections. Moreover, active immunization with the inactivated vaccines also conferred protection against homologous and heterologous infections in mice. Collectively, our results demonstrate the proof-of-concept for inactivated whole-virus based CVA10 vaccines.

Initiation and regulation of immune responses to immunization with whole inactivated vaccines prepared from two genetically and antigenically distinct lineages of Egyptian influenza A virus subtype H5N1.

Following the introduction of highly pathogenic avian influenza (HPAI) virus subtype H5N1, the Egyptian government implemented a massive poultry vaccination campaign as the cornerstone of its policies to control the virus. The efficacy of vaccination has been evaluated primarily by measuring titers of antibodies inhibiting the hemagglutinating activity of the viral hemagglutinin (HA). However, other aspects of the host response remain poorly understood. In the present study, in addition to hemagglutination inhibition (HI) titers, cytokine profiles were examined and IFNγ concentrations were measured in vivo after immunization with a whole inactivated virus (WIV) prepared from a classical strain of clade 2.2.1.2 (C121) and an antigenic drift variant of clade 2.2.1.1 (V1063). The results revealed an earlier response and higher HI titers and IFNγ levels in sera from chickens immunized with C121, accompanied by significantly higher expression of IL8, IL10, and IL18 in the spleen and IL6 and IL10 in the bursa, compared to those immunized with V1063. Furthermore, stimulation of the HD11 cell line with C121 induced gradual upregulation of pro-inflammatory cytokines, which was observed at 24 hours post-inoculation (hpi), and became more pronounced at 48 and 72 hpi, accompanied by upregulation of IFNα. Conversely, V1063 induced very early transient higher expression of pro-inflammatory cytokines at 3 and 6 hpi accompanied by upregulation of IL10, which then decreased at 24, 48 and 72 hpi. In summary, our results provide evidence of a correlation between adaptive immune responses induced by WIVs and higher expression of IL10 and IL18 in addition to early induction of IFNα. These findings could be used to improve immune responses induced by WIVs.

In Vivo Validation of Predicted and Conserved T Cell Epitopes in a Swine Influenza Model.

Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection include: inactivated whole-virus vaccines, subunit vaccines, and alpha replicon-based vaccines. As is true for influenza vaccines for humans, these strategies do not provide broad protection against the diverse strains of influenza A virus (IAV) currently circulating in U.S. swine. Improved approaches to developing swine influenza vaccines are needed. Here, we used immunoinformatics tools to identify class I and II T cell epitopes highly conserved in seven representative strains of IAV in U.S. swine and predicted to bind to Swine Leukocyte Antigen (SLA) alleles prevalent in commercial swine. Epitope-specific interferon-gamma (IFNγ) recall responses to pooled peptides and whole virus were detected in pigs immunized with multi-epitope plasmid DNA vaccines encoding strings of class I and II putative epitopes. In a retrospective analysis of the IFNγ responses to individual peptides compared to predictions specific to the SLA alleles of cohort pigs, we evaluated the predictive performance of PigMatrix and demonstrated its ability to distinguish non-immunogenic from immunogenic peptides and to identify promiscuous class II epitopes. Overall, this study confirms the capacity of PigMatrix to predict immunogenic T cell epitopes and demonstrate its potential for use in the design of epitope-driven vaccines for swine. Additional studies that match the SLA haplotype of animals with the study epitopes will be required to evaluate the degree of immune protection conferred by epitope-driven DNA vaccines in pigs.

Vaccine-associated enhanced respiratory disease is influenced by haemagglutinin and neuraminidase in whole inactivated influenza virus vaccines.

Multiple subtypes and many antigenic variants of influenza A virus (IAV) co-circulate in swine in the USA, complicating effective use of commercial vaccines to control disease and transmission. Whole inactivated virus (WIV) vaccines may provide partial protection against IAV with substantial antigenic drift, but have been shown to induce vaccine-associated enhanced respiratory disease (VAERD) when challenged with an antigenic variant of the same haemagglutinin (HA) subtype. This study investigated the role the immune response against HA, neuraminidase (NA) and nucleoprotein (NP) may play in VAERD by reverse engineering vaccine and challenge viruses on a common backbone and using them in a series of vaccination/challenge trials. Mismatched HA between vaccine and challenge virus was necessary to induce VAERD. However, vaccines containing a matched NA abrogated the VAERD phenomenon induced by the HA mismatch and this was correlated with NA-inhibiting (NI) antibodies. Divergence between the two circulating swine N2 lineages (92 % identity) resulted in a loss of NI cross-reactivity and also resulted in VAERD with the mismatched HA. The NP lineage selected for use in the WIV vaccine strains did not affect protection or pathology. Thus the combination of HA and NA in the vaccine virus strains played a substantial role in vaccine protection versus immunopathology, suggesting that vaccines that target the HA protein alone could be more prone to VAERD due to the absence of cross-protective NI antibodies.

Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies.

This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4+ T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value.

Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type

The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and respiratory specimens of pigs vaccinated with intramuscular whole-inactivated virus (WIV), intranasal live-attenuated influenza virus (LAIV), and intranasal wild type (WT) IAV. NI titers were also analyzed in sera from an investigation of piglet vaccination in the presence of passive maternally-derived antibodies. Test antigens contained genetically divergent swine-lineage NA genes homologous or heterologous to the vaccines with mismatched hemagglutinin genes (HA). Naïve piglets responded to WIV and LAIV vaccines and WT infection with strong homologous serum NI titers. Cross-reactivity to heterologous NAs depended on the degree of genetic divergence between the NA genes. Bronchoalveolar lavage specimens of LAIV and WT-immunized groups also had significant NI titers against the homologous antigen whereas the WIV group did not. Piglets of vaccinated sows received high levels of passive NI antibody, but their NI responses to homologous LAIV vaccination were impeded. These data demonstrate the utility of the enzyme-linked lectin assay for efficient NI antibody titration of serum as well as respiratory tract secretions. Swine IAV vaccines that induce robust NI responses are likely to provide broader protection against the diverse and rapidly evolving IAV strains that circulate in pig populations. Mucosal antibodies to NA may be one of the protective immune mechanisms induced by LAIV vaccines.