Inactivated Whole-virus Research Articles

Bursin-like peptide (BLP) enhances H9N2 influenza vaccine induced humoral and cell mediated immune responses

Vaccination with H9N2 avian influenza whole-inactivated virus (WIV) has been shown to be ineffective at eliciting sufficient humoral and cellular immunity against H9N2 avian influenza virus. This study assessed the effects of a synthetic Bursin-like epitope peptide (BLP) as adjuvant for H9N2 WIV in mice. Titers HI and avian influenza virus neutralizing antibodies, subtypes of HA antibodies, T helper (Th) cytokine levels, cytotoxic T-lymphocyte activities and changes in spleen T-cell subsets and natural killer cells were determined. We found that BLP induced a balance between IgG1 and IgG2a secretion levels. WIV antigen alone induced mainly Th1 cytokines secretion, whereas BLP showed increased secretion of Th1 and Th2 cytokines, including interleukin (IL)-2, interferon-γ (IFN-γ) and IL-4, but not IL-10, and may be resembles a Th0 like response. BLP significantly promoted growth and expansion of natural killer cells and of CD4+ and CD8+ T-cell subsets in the spleen. Meanwhile, BLP induced a better cytotoxic T-lymphocyte response to H9N2 virus. Furthermore, virus challenge experiments confirmed that BLP contributed to inhibition replication of the virus from mouse lungs. Taken together, these findings suggest that BLP may be an effective adjuvant for H9N2 avian influenza vaccine.

EV71 vaccine, an invaluable gift for children.

Enterovirus 71 (EV71) is a major pathogen for severe hand, foot and mouth disease (HFMD). Development of vaccines against EV71 would be the most effective approach to prevent the EV71 outbreak. Research and development (R&D) of EV71 vaccine was carried out in several Asian countries. Currently three companies in mainland China have completed Phase III clinical trials of inactivated EV71 whole-virus vaccines, whereas the other two companies have completed Phase I clinical trials separately in Taiwan and in Singapore. Results from those clinical trials have indicated high safety and immunogenicity of EV71 vaccine. Protective efficacies were over 90% on EV71-associated HFMD and over 80% on other EV71-associated diseases. In this paper, we summarize the results from three EV71 vaccine Phase III clinical trials and discuss the challenges of incorporating EV71 vaccine into Expanded Program on Immunization (EPI) in countries with EV71 epidemics.

Influenza A virus hemagglutinin protein subunit vaccine elicits vaccine-associated enhanced respiratory disease in pigs

Vaccine-associated enhanced respiratory disease (VAERD) can occur when pigs are challenged with heterologous virus in the presence of non-neutralizing but cross-reactive antibodies elicited by whole inactivated virus (WIV) vaccine. The aim of this study was to compare the effects of heterologous δ1-H1N2 influenza A virus (IAV) challenge of pigs after vaccination with 2009 pandemic H1N1 virus (H1N1pdm09) recombinant hemagglutinin (HA) subunit vaccine (HA-SV) or temperature-sensitive live attenuated influenza virus (LAIV) vaccine, and to assess the role of immunity to HA in the development of VAERD. Both HA-SV and LAIV vaccines induced high neutralizing antibodies to virus with homologous HA (H1N1pdm09), but not heterologous challenge virus (δ1-H1N2). LAIV partially protected pigs, resulting in reduced virus shedding and faster viral clearance, as no virus was detected in the lungs by 5 days post infection (dpi). HA-SV vaccinated pigs developed more severe lung and tracheal lesions consistent with VAERD following challenge. These results demonstrate that the immune response against the HA protein alone is sufficient to cause VAERD following heterologous challenge.

Divergent immune responses and disease outcomes in piglets immunized with inactivated and attenuated H3N2 swine influenza vaccines in the presence of maternally-derived antibodies

Live-attenuated influenza virus (LAIV) prime-boost vaccination previously conferred protection against heterologous H3N2 swine influenza challenge, including in piglets with maternally derived antibodies (MDA). Conversely, a whole-inactivated virus (WIV) vaccine was associated with enhanced disease. This study was aimed at identifying immune correlates of cross-protection. Piglets with and without MDA received intramuscular adjuvanted WIV or intranasal LAIV, and were challenged with heterologous H3N2. WIV induced cross-reactive IgG, inhibited by MDA, and a moderate T cell response. LAIV elicited mucosal antibodies and T cells cross-reactive to the heterologous challenge strain. The presence of MDA at LAIV vaccination blocked lung and nasal antibody production, but did not interfere with T cell priming. Even without mucosal antibodies, MDA-positive LAIV vaccinates were protected, indicating a likely role for T cells. Based on the data, one LAIV dose can induce cell-mediated immunity against antigenically divergent H3N2 influenza virus despite passive antibody interference with humoral immune responses.

Cross-fostering to prevent maternal cell transfer did not prevent vaccine-associated enhanced respiratory disease that occurred following heterologous influenza challenge of pigs vaccinated in the presence of maternal immunity.

Whole inactivated virus (WIV) vaccines for influenza A virus (IAV) provide limited cross-protection to diverse antigenic strains that are circulating or may emerge in a population. Maternal vaccination is used to protect neonatal animals from disease through passive transfer of immunity. It is desirable to vaccinate at a young age to induce active immunity that provides protection against infection before maternal immunity wanes. However, maternal-derived immunity (MDI; antibody or cells) can interfere with vaccine priming. Previous work indicates that vaccine-associated enhanced respiratory disease (VAERD) occurs in pigs following heterologous IAV challenge if pigs were previously vaccinated with WIV vaccine in the presence of matched MDI. However, the component of MDI (antibody or cells) that is required for the mispriming of piglet immunity has not been determined. While antibody from colostrum is absorbed into piglet circulation regardless of the sow from which it receives colostrum, transfer of maternal cells requires colostrum from the biological dam. We used cross-fostering (CF) as a tool to determine if maternal cells are required for the mispriming of piglet immunity upon WIV vaccination in the presence of MDI. Piglets vaccinated in the presence of MDI, regardless of CF, displayed characteristics of VAERD following heterologous challenge. MDI alone (no piglet vaccination) did not provide cross-protection against the antigenic variant. However, it did not induce VAERD. WIV vaccination provided complete protection against homologous challenge when delivered to piglets without MDI. Vaccination in the presence of MDI inhibited an increase in hemagglutination inhibiting (HI) antibody titers to vaccine antigen, but did not alter development of total immunoglobulin levels to vaccine virus. Taken together, the cellular component of MDI did not contribute to the mispriming of piglet immunity to WIV vaccine, but maternal-derived antibody (MDA) alone was sufficient. Future work is aimed at understanding how MDA alters WIV vaccine immunogenicity.

A single dose of whole inactivated H7N9 influenza vaccine confers protection from severe disease but not infection in ferrets

The H7N9 influenza virus caused significant mortality and morbidity in infected humans during an outbreak in China in 2013 stimulating vaccine development efforts. As previous H7-based vaccines have been poorly immunogenic in humans we sought to determine the immunogenic and protective properties of an inactivated whole virus vaccine derived from a 2013 H7N9 virus in ferrets. As whole virus vaccine preparations have been shown to be more immunogenic in humans, but less likely to be used, than split or surface antigen formulations, we vaccinated ferrets with a single dose of 15, 30, or 50μg of the vaccine and subsequently challenged with wild-type A/Anhui/1/2013 (H7N9) either by direct instillation or by contact with infected animals. Although ferrets vaccinated with higher doses of vaccine had higher serum hemagglutinin inhibition (HI) titers, the titers were still low. During subsequent instillation challenge, however, ferrets vaccinated with 50μg of vaccine showed no illness and shed significantly less virus than mock vaccinated controls. All vaccinated ferrets had lower virus loads in their lungs as compared to controls. In a separate study where unvaccinated-infected ferrets were placed in the same cage with vaccinated-uninfected ferrets, vaccination did not prevent infection in the contact ferrets, although they showed a trend of lower viral load. Overall, we conclude that inactivated whole-virus H7N9 vaccine was able to reduce the severity of infection and viral load, despite the lack of hemagglutinin-inhibiting antibodies.

Mucosal correlates of cross-protection for live-attenuated influenza virus vaccines in pigs (VET2P.1031)

Abstract Controlling swine influenza A virus (IAV) has become increasingly difficult with the emergence of novel reassorted strains and introduction of human seasonal IAV into pigs. In North American swine there are 6 antigenically distinct H1 subtypes currently circulating. Live-attenuated influenza vaccines (LAIV) provide broader cross-protection than whole-inactivated virus (WIV) vaccine making LAIV a candidate for next-generation swine IAV vaccines. However, there is no standardized assay to predict cross-protection following LAIV vaccination. Hemagglutination-inhibiting (HI) immunoglobulin (Ig) in serum has long been the gold standard correlate of protection following WIV vaccination; however, LAIV does not elicit a robust serum HI Ig titer. Oral fluids (OF) have become a rapidly developing diagnostic specimen for a variety of animal pathogens. We evaluated nasal wash (NW) and OF samples for measuring cross-reactive Ig responses to a panel of H1 viruses following vaccination. Both NW and OF had detectable levels of IAV-specific Ig when measured by ELISA. NW from LAIV vaccinated pigs had significant cross-reactive IgA titers, while NW from WIV vaccinated pigs did not have detectable IgA. Samples of OF from either LAIV or WIV vaccinated pigs had high titers of cross-reactive IAV-specific IgG and measurable IgA. OF samples also displayed IAV specific neutralization. These data suggest OF can serve as a sample for evaluating LAIV immunogenicity and predicting cross-protection.

Specificities of human CD4+ T cell responses to an inactivated flavivirus vaccine and infection: correlation with structure and epitope prediction.

Tick-borne encephalitis (TBE) virus is endemic in large parts of Europe and Central and Eastern Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to the mosquito-borne yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated whole-virus vaccine can effectively prevent clinical disease. Neutralizing antibodies are directed to the viral envelope protein (E) and an accepted correlate of immunity. However, data on the specificities of CD4(+) T cells that recognize epitopes in the viral structural proteins and thus can provide direct help to the B cells producing E-specific antibodies are lacking. We therefore conducted a study on the CD4(+) T cell response against the virion proteins in vaccinated people in comparison to TBE patients. The data obtained with overlapping peptides in interleukin-2 (IL-2) enzyme-linked immunosorbent spot (ELISpot) assays were analyzed in relation to the three-dimensional structures of the capsid (C) and E proteins as well as to epitope predictions based on major histocompatibility complex (MHC) class II peptide affinities. In the C protein, peptides corresponding to two out of four alpha helices dominated the response in both vaccinees and patients, whereas in the E protein concordance of immunodominance was restricted to peptides of a single domain (domain III). Epitope predictions were much better for C than for E and were especially erroneous for the transmembrane regions. Our data provide evidence for a strong impact of protein structural features that influence peptide processing, contributing to the discrepancies observed between experimentally determined and computer-predicted CD4(+) T cell epitopes. Importance: Tick-borne encephalitis virus is endemic in large parts of Europe and Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated vaccine can effectively prevent disease. Both vaccination and natural infection induce the formation of antibodies to a viral surface protein that neutralize the infectivity of the virus and mediate protection. B lymphocytes synthesizing these antibodies require help from other lymphocytes (helper T cells) which recognize small peptides derived from proteins contained in the viral particle. Which of these peptides dominate immune responses to vaccination and infection, however, was unknown. In our study we demonstrate which parts of the proteins contribute most strongly to the helper T cell response, highlight specific weaknesses of currently available approaches for their prediction, and demonstrate similarities and differences between vaccination and infection.

Suramin inhibits EV71 infection

Enterovirus-71 (EV71) is one of the major causative reagents for hand-foot-and-mouth disease. In particular, EV71 causes severe central nervous system infections and leads to numerous dead cases. Although several inactivated whole-virus vaccines have entered in clinical trials, no antiviral agent has been provided for clinical therapy. In the present work, we screened our compound library and identified that suramin, which has been clinically used to treat variable diseases, could inhibit EV71 proliferation with an IC50 value of 40μM. We further revealed that suramin could block the attachment of EV71 to host cells to regulate the early stage of EV71 infection, as well as affected other steps of EV71 life cycle. Our results are helpful to understand the mechanism for EV71 life cycle and provide a potential for the usage of an approved drug, suramin, as the antiviral against EV71 infection.

Immunologic characterization of a novel inactivated nasal mumps virus vaccine adjuvanted with Protollin

An inactivated, mucosal mumps virus (MuV) vaccine would address many of the problems associated with current live-attenuated formulations. Protollin (Prl)-based adjuvants (containing TLR2 and TLR4 ligands) are well-suited for nasal administration. We sought to develop an inactivated whole-virus nasal vaccine for MuV using the Prl adjuvant/delivery vehicle and to test tolerability and immunogenicity in a mouse model. BALB/c mice exhibited signs of transient reactogenicity (hunched posture, erect fur, weight loss ≤10% of total body weight) following administration of intranasal MuV-Prl vaccines, though most of these manifestations resolved within 24h. Compared to high-dose unadjuvanted vaccine (8μgMuV), administration of high-dose adjuvanted formulation (8μgMuV-Prl) induced greater MuV-specific serum IgG (3.26E6ng/mL vs. 2.2E5ng/mL, 8μgMuV-Prl vs. 8μgMuV, p<0.001) and mucosal IgA (128ng/mL vs. 45ng/mL, 8μgMuV-Prl vs. 8μgMuV, p<0.05). Serum IgG isotypes and splenocyte cytokine secretion induced by MuV-Prl suggested a predominant T helper cell (Th)1-type immune response. This response was characterized by: (1) ≥four-fold increase of IgG2a levels compared to IgG1; and (2) high IL-2 (644pg/mL)/IFN-γ (228pg/mL) and low IL-5 (31pg/mL) secretion in MuV-restimulated splenocytes from animals receiving MuV-Prl formulations. MuV-Prl vaccination induced higher levels of serum antibodies capable of neutralizing MuV in vitro than MuV alone, particularly for high-dose 8μg formulations (357 neutralizing units (NU)/mL vs. 32NU/mL, 8μgMuV-Prl vs. 8μgMuV, p<0.001). Thus, nasal MuV-Prl vaccines are fairly well-tolerated and highly immunogenic in mice.

Swine influenza virus vaccine serologic cross‐reactivity to contemporary US swine H3N2 and efficacy in pigs infected with an H3N2 similar to 2011–2012 H3N2v

Swine influenza A virus (IAV) reassortment with 2009 H1N1 pandemic (H1N1pdm09) virus has been documented, and new genotypes and subclusters of H3N2 have since expanded in the US swine population. An H3N2 variant (H3N2v) virus with the H1N1pdm09 matrix gene and the remaining genes of swine triple reassortant H3N2 caused outbreaks at agricultural fairs in 2011-2012. To assess commercial swine IAV vaccines' efficacy against H3N2 viruses, including those similar to H3N2v, antisera to three vaccines were tested by hemagglutinin inhibition (HI) assay against contemporary H3N2. Vaccine 1, with high HI cross-reactivity, was further investigated for efficacy against H3N2 virus infection in pigs with or without maternally derived antibodies (MDA). In addition, efficacy of a vaccine derived from whole inactivated virus (WIV) was compared with live attenuated influenza virus (LAIV) against H3N2. Hemagglutinin inhibition cross-reactivity demonstrated that contemporary swine H3N2 viruses have drifted from viruses in current swine IAV vaccines. The vaccine with the highest level of HI cross-reactivity significantly protected pigs without MDA. However, the presence of MDA at vaccination blocked vaccine efficacy. The performance of WIV and LAIV was comparable in the absence of MDA. Swine IAV in the United States is complex and dynamic. Vaccination to minimize virus shedding can help limit transmission of virus among pigs and people. However, vaccines must be updated. A critical review of the use of WIV in sows is required in the context of the current IAV ecology and vaccine application in pigs with MDA.

Antigenic analysis of H5N1 highly pathogenic avian influenza viruses circulating in Egypt (2006–2012)

The highly pathogenic avian influenza (HPAI) H5N1 in Egypt circulated continuously after its introduction in February 2006 with substantial economic losses and frequent human infections. Phylogenetic analysis of the available HA sequences revealed the presence of two main sublineages; the classic 2.2.1 and the variant 2.2.1.1. The classic 2.2.1 had subdivided into two clusters of viruses; cluster C1 contained the originally introduced virus and isolates from 2006 to 2009 and cluster C2 emerged in 2007 and continues to circulate. The variant 2.2.1.1 represents the isolates mainly from chickens and subdivided into two clusters; cluster V1 contains isolates from 2007 to 2009 and cluster V2 contains isolates from 2008 to 2011. Sequence analysis revealed 28 amino acid mutations in the previously reported antigenic sites and high evolution rate which may be due to selective pressure from vaccination and/or natural infection. Antigenic analysis of 18 H5N1 isolates from 2006 to 2012 that represent different clusters was conducted using hemagglutination inhibition (HI) and virus neutralization (VN) assays using hyperimmune sera produced by immunizing SPF chickens with inactivated whole-virus. Antigenic relatedness of ancestral Egyptian H5N1 isolate (459-3/06) with other isolates ranged from 30.7% to 79.1% indicating significant antigenic drift of the H5N1 viruses from the ancestral strains. The antigenic relatedness between C2 and V2 clusters ranged from 28.9% to 68% supporting the need for vaccine seed strains from both clusters. Interestingly, A/CK/EG/1709-6/2008 H5N1 strain showed a broad cross reactivity against viruses in different H5N1 clusters (antigenic relatedness ranged from 63.9% to 85.8%) demonstrating a potential candidate as a vaccine strain. Antigenic cartography which facilitates a quantitative interpretation and easy visualization of serological data was constructed based on HI results and further demonstrated the several antigenic groups among Egyptian H5N1 viruses. In conclusion, the cross reactivity between the co-circulating H5N1 strains may not be adequate for protection against each other and it is recommended to test vaccines that contain isolates from different antigenic groups in experimental infection trials for the selection of vaccine seed strain. Furthermore, the continuous monitoring for detecting the emerging variants followed by detailed antigenic analysis for updating vaccines is warranted.

Heterosubtypic cross‐protection induced by whole inactivated influenza virus vaccine in mice: influence of the route of vaccine administration

Development of influenza vaccines capable of inducing broad protection against different virus subtypes is necessary given the ever-changing viral genetic landscape. Previously, we showed that vaccination with whole inactivated virus (WIV) induces heterosubtypic protection against lethal virus infection in mice. Whole inactivated virus-induced cross-protection was found to be mediated primarily by flu-specific CD8+ T cells. As it has been demonstrated that the route of vaccine administration strongly influences both the quantity and quality of vaccine-induced immunity, in this study, we determined which route of WIV administration induces optimal heterosubtypic cross-protection. We compared the magnitude of the immune response and heterosubtypic protection against lethal A/PR/8/34 (H1N1) infection after subcutaneous (SC), intramuscular (IM), and intranasal (IN) vaccination with A/NIBRG-14 (H5N1) WIV. Subcutaneous and IM administration was superior to IN administration of influenza WIV in terms of flu-specific CD8+ T-cell induction and protection of mice against lethal heterosubtypic challenge. Surprisingly, despite the very low flu-specific CD8+ T-cell responses detected in IN-vaccinated mice, these animals were partially protected, most likely due to cross-reactive IgA antibodies. The results of this study show that the magnitude of WIV-induced flu-specific CD8+ T-cell activity depends on the applied vaccination route. We conclude that parenteral administration of WIV vaccine, in particular IM injection, is superior to IN vaccine delivery for the induction of heterosubtypic cross-protection and generally appears to elicit stronger immune responses than mucosal vaccination with WIV.

Critical Role of TLR7 Signaling in the Priming of Cross-Protective Cytotoxic T Lymphocyte Responses by a Whole Inactivated Influenza Virus Vaccine

Current influenza vaccines fail to induce protection against antigenically distinct virus strains. Accordingly, there is a need for the development of cross-protective vaccines. Previously, we and others have shown that vaccination with whole inactivated virus (WIV) induces cross-protective cellular immunity in mice. To probe the mechanistic basis for this finding, we investigated the role of TLR7, a receptor for single-stranded RNA, in induction of cross-protection. Vaccination of TLR7−/− mice with influenza WIV failed to protect against a lethal heterosubtypic challenge; in contrast, wild-type mice were fully protected. The lack of protection in TLR7−/− mice was associated with high viral load and a relative paucity of influenza-specific CD8+ cytotoxic T lymphocyte (CTL) responses. Dendritic cells (DCs) from TLR7−/− mice were unable to cross-present WIV-derived antigen to influenza-specific CTLs in vitro. Similarly, TLR7−/− DCs failed to mature and become activated in response to WIV, as determined by the assessment of surface marker expression and cytokine production. Plasmacytoid DCs (pDCs) derived from wild-type mice responded directly to WIV while purified conventional DCs (cDCs) did not respond to WIV in isolation, but were responsive in mixed pDC/cDC cultures. Depletion of pDCs prior to and during WIV immunization resulted in reduced numbers of influenza-specific CTLs and impaired protection from heterosubtypic challenge. Thus, TLR7 plays a critical role in the induction of cross-protective immunity upon vaccination with WIV. The initial target cells for WIV appear to be pDCs which by direct or indirect mechanisms promote activation of robust CTL responses against conserved influenza epitopes.

A pathogenic picornavirus acquires an envelope by hijacking cellular membranes

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane1, attributes that profoundly affect stability, transmission, and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically-transmitted hepatitis2–4. HAV infects in a stealth-like manner and replicates efficiently in the liver5. Virus-specific antibodies appear only after 3–4 weeks of infection, and typically herald its resolution3,4. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure6, when virus replication is well established in the liver5. Here, we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (“eHAV”) resemble exosomes7, small vesicles that are increasingly recognized to play important roles in intercellular communications. They are fully infectious, sensitive to chloroform extraction, and circulate in the blood of infected humans. Their biogenesis is dependent upon host proteins associated with endosomal-sorting complexes required for transport (ESCRT)8, VPS4B and ALIX. While the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and likely promotes virus spread within the liver, anti-capsid antibodies restrict replication following infection with eHAV, suggesting a possible explanation for post-exposure prophylaxis. Membrane hijacking by HAV blurs the classic distinction between “enveloped” and “nonenveloped” viruses, and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

A Phase I, randomized, open-label study to evaluate the safety and immunogenicity of an enterovirus 71 vaccine

BackgroundLarge-scale outbreaks of enterovirus 71 (EV71) infections have occurred in Asia-Pacific regions. Severe complications include encephalitis and poliomyelitis-like paralysis, cardiopulmonary collapse, and death, necessitating an effective vaccine against EV71. MethodsIn this randomized Phase I study, we evaluated the safety and immunogenicity of an inactivated alum-adjuvanted EV71 whole-virus vaccine produced on Vero cell cultures. Sixty healthy volunteers aged 20–60 years received two doses of vaccine, administered 21 days apart. Each dose contained either 5μg of EV71 antigen with 150μg of adjuvant (Group A05) or 10μg of EV71 antigen with 300μg of adjuvant (Group B10). Serologic analysis was performed at baseline, day 21, and day 42. ResultsThere were no serious adverse events. Mild injection site pain and myalgia were the most common adverse events with either vaccine formulation. The immunogenicity data showed that 90% of vaccine recipients have a 4-fold or greater increase in neutralization antibody titers (NT) after the first dose, without a further increase in NT after the second dose. The seroconversion rates on day 21 and day 42 were 86.7% and 93.1% respectively, in Group A05, and 92.9% and 96.3%, respectively, in Group B10. Thus, 5μg and 10μg of the EV71 vaccine can induce a remarkable immune response in healthy adults after only the first vaccination. ConclusionThe 5μg and 10μg adjuvanted EV71 vaccines are generally safe and immunogenic in healthy adults. (ClinicalTrials.gov number, NCT01268787).

Identification of conserved neutralizing linear epitopes within the VP1 protein of coxsackievirus A16

Coxsackievirus A16 (CA16) is a major causative agent of hand, foot, and mouth disease. Immunization with inactivated whole-virus or recombinant virus-like particles (VLP) of CA16 elicits neutralizing antibodies that protect mice against lethal challenge. However, the epitope/s responsible for this induction has not been determined. In this investigation, we identified six neutralizing linear epitopes of CA16. A panel of 95 synthetic peptides spanning the entire VP1 protein of CA16 were screened by ELISA for reactivity with neutralizing antisera against CA16 VLPs, which were generated in a previous study (Vaccine 30:6642–6648). Fifteen high-binding peptides were selected and further examined for their inhibitory effect on neutralization by anti-VLP sera. Among them, six peptides with no overlap significantly inhibited neutralization. Mice immunized with these six peptides generated peptide-specific serum antibodies. The anti-peptide antisera positively detected CA16 via immunofluorescent staining and Western blot assays. More importantly, they neutralized both homologous and heterologous CA16 strains, indicating that these six peptides represented neutralizing epitopes. Sequence alignment also showed that these epitopes are extremely conserved among CA16 strains of different genotypes. These findings have important implications for the development of peptide-based broadly protective CA16 vaccines.

Active immunization with a Coxsackievirus A16 experimental inactivated vaccine induces neutralizing antibodies and protects mice against lethal infection

Coxsackievirus A16 (CA16) is one of the main pathogens that cause hand, foot and mouth disease, which frequently occurs in young children. A small percentage of patients infected with CA16 may suffer from severe neurological complications that could also lead to death. Recent epidemiological data shows the increase in both the total number and the incidence rate of severe CA16-associated cases in China, indicating that CA16 should be targeted for vaccine development. In this article, we report the immunogenicity and protective efficacy of experimental inactivated CA16 vaccines in mice. We show that immunization with β-propiolactone-inactivated whole-virus vaccines derived from two CA16 clinical isolates were able to induce CA16-specific antibody and IFN-secreting T-cell responses in mice. The resulting anti-CA16 mouse sera neutralized both homologous and heterologous CA16 clinical isolates, as well as a mouse-adapted strain called CA16-MAV, which is capable of infecting 14-day-old mice. Passive transfer of anti-CA16 neutralizing sera partially protected neonatal mice from lethal challenge by a clinical isolate CA16-G08. More significantly, active immunization of mice with the inactivated vaccines conferred complete protection against lethal infection with CA16-MAV. Collectively, these results provide a solid foundation for further development of inactivated whole-virus CA16 vaccines for human use.

Comparison of protection against H5N1 influenza virus in mouse offspring provided by maternal vaccination with HA DNA and inactivated vaccine

H5N1 influenza virus is one of the viruses that can potentially cause an influenza pandemic. Protection of newborns against influenza virus infection could be effectively provided by maternal immunization. In this study, female mice were immunized with H5N1 HA DNA vaccine or inactivated whole-virion vaccine, and the protection provided by maternal antibodies in their offspring against a lethal homologous influenza virus challenge was compared. The results showed that maternal antibodies, whether induced by a DNA vaccine or an inactivated vaccine, could completely protect offspring aged 1-4 weeks from a lethal influenza virus challenge. Breast-feeding was the major route of transfer for maternal antibodies. Milk-derived antibodies were able to effectively protect the offspring aged 1-4 weeks from lethal influenza virus infection, whereas maternal antibodies transferred through the placenta only partially protected the offspring 1-2 weeks of age. The milk- and placenta-transferred IgG2a antibody levels in offspring from their mothers, whether vaccinated with DNA vaccine or inactivated vaccine, were higher than the IgG1 levels. Our results indicated that maternal vaccination with HA DNA, as well as with whole-virion inactivated vaccine, could offer effective protection to offspring against H5N1 influenza virus infection.

Preliminary Assessment of Safety and Immunogenicity of an Inactivated Whole-Virion Vaccine against Influenza А (H1N1) Pdm09 Containing Aluminum Hydroxide Adjuvant: A Randomized, Blinded Phase I Clinical Study

We performed a randomized, blinded, dose-dependent placebo-controlled phase I clinical study of single administration of Refluvac®, a monovalent inactivated whole-virion vaccine against pandemic influenza А (Н1N1) pdm09 containing aluminum adjuvant, in healthy volunteers aged 18-60. Single intramuscular injection at doses of 3.75, 7.5, or 15.0 µg hemagglutinin (НА) identified no safety issues in adult volunteers ( n=12 per group); no severe or serious vaccination-related adverse events were observed. Only mild/moderate local adverse events (n= 3/12, 25% in 7.5 µg НА arm) and one moderate systemic reaction ( n=1/12, 8.3% in 15.0 µg НА arm) were observed. In volunteers vaccinated at 3.75 µg HA, the proportion of subjects with 4-fold seroconversion was 75%, the level of seroprotection was also 75%, the antibody titer increase was 10.7-fold, and the geometric mean titer (GMT) of antibodies to А (H1N1) pdm09 was 53.4; for the 7.5 µg HA dose, the proportion of 4-fold seroconversion was 75%, the antibody titer increase was 32.0-fold, the GMT was 160.0, and the level of seroprotection was 75%. When administered at a higher dose (15 µg HA), the proportion of subjects with protective antibody titers increased from 75% to 83%; however, the GMT and antibody titer increase were not significantly different (P>0.05) to the group vaccinated at 7.5 µg HA. Phase II clinical studies of the 3.75 and 7.5 µg HA doses of Refluvac® vaccine should be performed in a larger cohort of healthy volun teers aged 18-60. The effective immunogenicity of low doses of Refluvac® vaccine may enable increased production of pandemic influenza vaccines, and thus provide more people with a safe, ef fective vaccine.