Evaluation of a vectored equine herpesvirus type 1 (EHV-1) vaccine expressing H3 haemagglutinin in the protection of dogs against canine influenza.

In 2004, canine influenza virus (CIV) was identified as a respiratory pathogen of dogs for the first time and found to be closely related to H3N8 equine influenza virus (EIV). We generated a recombinant vectored vaccine that expresses H3 of a recent isolate of EIV using equine herpesvirus type 1 (EHV-1) as the delivery vehicle. This EHV-1 vectored vaccine exhibited robust and stable EIV H3 expression and induced a strong influenza virus-specific response in both mice and dogs upon intranasal or subcutaneous administration. Furthermore, upon challenge with the recent CIV isolate A/canine/PA/10915-07, protection of vaccinated dogs could be demonstrated by a significant reduction in clinical sings, and, more importantly, by a significant reduction in virus shedding. We concluded that the EHV-1/H3 recombinant vector can be a valuable alternative for protection of dogs against clinical disease induced by CIV and can significantly reduce virus spread.     

Efficacy of a vaccine that links viral epitopes to flagellin in protecting aged mice from influenza viral infection

Influenza vaccines are less effective in older people than younger people. This impaired ability to protect older people from influenza viral lung infection has important implications as older people suffer a higher morbidity and mortality from influenza viral lung infection than younger people. Therefore, the development of novel effective vaccines that induce protection from influenza viral infections in older people are urgently needed. We had previously shown that direct linking the TLR5 activator, flagellin, to viral peptides induces effective immunity to viral antigens in young mice and people, respectively. In this study, we tested the efficacy of this vaccine platform with the hemagglutinin peptide of the influenza A strain virus (vaccine denoted as STF2.HA1-2) in protecting aged mice from subsequent influenza viral lung infection. We found that a 3.0 μg dose of the vaccine was effective in reducing mortality and increasing clinical well-being during influenza viral lung infection in aged mice. However, this effect was inferior to the response induced in young mice. Defects in the adaptive immune system but not the innate immune system were associated with this reduced effectiveness of the vaccine with aging. Our results indicate that the STF2.HA1-2 vaccine is effective in protecting aged hosts from influenza lung infection, although defects in the adaptive immune system with aging may limit the effectiveness of this vaccine in older people.     

A recombinant rabies vaccine expressing the trimeric form of the glycoprotein confers enhanced immunogenicity and protection in outbred mice

Rabies is a disease characterized by an invariably lethal encephalitis of viral origin that can be controlled by preventive vaccination programs of wildlife, domestic animals and humans in areas with a high risk of exposure. Currently available vaccines are expensive, cumbersome to produce and require intensive immunization and booster schemes to induce and maintain protective immunity. In the present study, we describe the development of candidate recombinant subunit rabies vaccines based on the glycoprotein G of the prototype rabies virus (RABV-G) expressed either as a monomer (RABV-mG) or in its native trimeric configuration (RABV-tG), with or without Matrix-M™ adjuvant. Immunogenicity and protective efficacy of the respective candidate vaccines were tested in outbred NIH Swiss albino mice. The RABV-tG candidate vaccine proved to be superior to the RABV-mG vaccine candidate both in terms of immunogenicity and efficacy. The relatively poor immunogenicity of the RABV-mG vaccine candidate was greatly improved by the addition of the adjuvant. A single, low dose of RABV-tG in combination with Matrix-M™ induced high levels of high avidity neutralizing antibodies and protected all mice against challenge with a lethal dose of RABV. Consequently RABV-tG used in combination with Matrix-M™ is a promising vaccine candidate that overcomes the limitations of currently used vaccines.     

Influenza vaccine strain selection: equivalence of two antigenically distinct haemagglutinin variants of 1989 H3N2 influenza A virus in protection of mice.

Precise antigenic analysis with haemagglutinin-inhibition (HI) tests of 1989 H3N2 influenza A viruses with polyclonal ferret, rabbit and mouse antisera has shown, first, significant differences among 1989 wild-type isolates, second, antigenic differences between two high-yield vaccine candidate reassortant viruses, third, significant antigenic differences of one reassortant (X-105) from the wild-type virus (A/Guangdong A/39) from which it was derived, and fourth, dependence of antigenic characterization of viruses upon the host species used in immunization. Nevertheless, the two reassortant viruses (only 43% similar by HI test) were equally protective in preventing homovariant or heterovariant infection in either previously unimmunized or infection-primed mice. These results not only confirm the known antigenic heterogeneity of influenza A viruses, but raise questions about the adequacy of current methods of antigenic characterization of influenza viruses and the basis for decisions on vaccine strain selection.     

A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.     

Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin

Immunization of ferrets with a plasmid DNA expressing influenza virus haemagglutinin (pCMV/H1 DNA) provided complete protection from challenge with the homologous A/PR/8/34 (HINI) influenza virus. Delivery of DNA-coated gold beads by gene gun to the epidermis was much more efficient than intramuscular delivery of DNA in aqueous solution. The antibody response induced by DNA delivered by gene gun was more cross-reactive than DNA delivered in aqueous solution or after natural infection. This novel approach to vaccination against influenza may afford broader protection against antigenic drift than that provided by natural infection.     

A surface antigen influenza vaccine. 1. Purification of haemagglutinin and neuraminidase proteins.

Influenza virus was centrifuged in a KII rotor through a sucrose gradient containing Triton N101, a non-ionic surfactant. The micelles of surfactant formed a band in the gradient. As virus particles passed through the surfactant, the haemagglutinin and neuraminidase proteins were stripped from the surface and remained near the surfactant micelles. The residual virus particles sedimented into a denser region of the gradient and were thus separated from the haemagglutinin and neuraminidase antigens. Fractions containing the surface antigens were pooled and Triton was removed by phase-separation at the cloud point.     

Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice

The replicon of Semliki Forest virus (SFV) offers the possibility to direct high-level, transient expression of heterologous proteins in vivo. We initiated studies to determine the possibility of employing the SFV expression system for recombinant vaccine purposes. Mice immunized with recombinant SFV encoding Influenza A nucleoprotein (NP) or E. coli LacZ developed long-lasting antigen-specific IgG levels and induction of cytotoxic T-cell (CTL) memory that persisted for over one year. Predominantly type 1 T-helper cells were induced as shown by IgG subclass ELISA. Humoral and cell-mediated immune responses could be induced upon delivery by several administration routes and mucosal immunizations induced secretory IgA in the respiratory tract. Development of immune responses against the vector itself did not inhibit boost responses by subsequent immunizations with recombinant SFV. Immunization of mice with vectors encoding the Influenza A virus antigens nucleoprotein (NP) and hemagglutinin (HA) resulted in immune responses that were protective against challenge infection with Influenza virus.     

Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin

The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family is critical for the activation of the adaptive immune response. Thus, incorporation of PAMPs into vaccines should result in more potent, protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we describe an influenza A vaccine that is refractory to the genetic instability of hemagglutinin and neuraminidase and includes a trigger of the innate immune response to enhance immunogenicity and efficacy. A recombinant protein comprising the TLR5 ligand flagellin fused to four tandem copies of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli and purified to homogeneity. This protein, STF2.4xM2e, retained TLR5 activity and displayed the protective epitope of M2e defined by a monoclonal antibody, 14C2. Mice immunized with STF2.4xM2e in aqueous buffer, without adjuvants or other formulation additives, developed potent M2e-specific antibody responses that were quantitatively and qualitatively superior to those observed with M2e peptide delivered in alum. The antibody response was dependent on the physical linkage of the antigen to flagellin and recognized the epitope defined by monoclonal antibody 14C2, which has been shown to protect mice from challenge with influenza A virus. Moreover, immunization with STF2.4xM2e at a dose of 0.3 microg per mouse protected mice from a lethal challenge with influenza A virus, and significantly reduced weight loss and clinical symptoms. These data demonstrate that the linkage of specific TLR ligand with influenza M2e yields a vaccine candidate that offers significant promise for widespread protection against multiple influenza A virus strains.     

A ferritin nanoparticle vaccine for foot-and-mouth disease virus elicited partial protection in mice

Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious infectious disease common in cloven-hoofed animals. Outbreaks and epidemics of FMD can result in major economic losses of livestock. Using ferritin nanoparticles as the scaffold for an antigen can enhance the immunogenicity of the subunit vaccine and provide possible protection against FMD. We used a baculovirus expression system to express four recombinant proteins (VP1, VP1-Ft, G-H loop-Ft, and ferritin) and the protective immunity of the FMD ferritin nanoparticle vaccines was evaluated in mice. The recombinant subunit vaccines containing VP1, VP1-Ft, and G-H loop-Ft proteins significantly increased FMDV-specific IgG and IgG subclass antibody titers compared with the PBS group, as well as enhancing splenocyte proliferation and the expression of IL-4 and IFN-γ. The VP1 and VP1-Ft vaccines provided survival rates of 55.6% and 66.7%, respectively. The G-H loop-Ft vaccine provided a 77.8% survival rate compared with 100% survival in the inactivated vaccine group. The partial survival provided by the ferritin nanoparticle vaccines indicated that further study of the effects of the fused ferritin nanoparticle FMDV vaccines in animals is warranted.     

Intramuscular immunization with a vesicular stomatitis virus recombinant expressing the influenza hemagglutinin provides post-exposure protection against lethal influenza challenge

Vaccines currently licensed for the prevention of seasonal influenza induce antibodies against the influenza hemagglutinin (HA) and neuraminidase (NA) contained in the vaccine preparation but require at least 2 weeks after immunization for the development of protective immunity. These vaccines do not induce protective responses quickly enough to blunt the effects of infection when administered after exposure. We have developed a novel vaccine based on recombinant vesicular stomatitis virus which expresses the influenza hemagglutinin (rVSV HA) and protects mice from lethal influenza challenge when the vaccine is administered intramuscularly at least 24 h after delivery of the influenza challenge virus. To our knowledge this is the first vaccine that effectively protects animals from lethal influenza challenge when delivered by a systemic route after influenza exposure has occurred. The induction of HA-specific immune responses by the vaccine is necessary for full protection from challenge, because animals immunized with an empty rVSV vector were not protected equally. Our results are consistent with a model in which vaccination induces an immediate antiviral cytokine response, followed by development of humoral and cellular immune responses which act to reduce pulmonary viral loads and accelerate recovery. Consistent with this model, mice vaccinated with the specific vaccine rVSV HA had high levels of IFN-α in the serum by 24 h after challenge/vaccination, developed serum neutralizing Ab to influenza 2 days prior to control animals, and had detectable anti-HA CD8 T cells present in the peripheral blood 3 days prior to control mice.     

Safety of recombinant quadrivalent influenza vaccine compared to inactivated influenza vaccine in Chinese adults: An observational study

BackgroundRecombinant influenza vaccine (RIV) has been in use in US adults since 2013. This study evaluated the safety of quadrivalent recombinant influenza vaccine (RIV4, Flublok® Quadrivalent, Sanofi Pasteur) compared with standard-dose quadrivalent inactivated influenza vaccine (SD-IIV4) in self-identified Chinese adults at Kaiser Permanente Northern California (KPNC).MethodsThis study evaluated adults aged 18–64 years within KPNC during the 2018–2019 influenza season who self-identified as Chinese (NCT03694392). We compared the rates of prespecified diagnoses of interest in the emergency department and inpatient settings as done in prior influenza studies, for three risk intervals: 0–2 days, 0–13 days, and 0–41 days following influenza vaccination, as well as number of deaths within 0–180 days after vaccination. We estimated the odds ratios (ORs) and 95% confidence intervals using logistic regression adjusted for sex, age group, presence of comorbidities, and same-day concomitant vaccination.ResultsComparing 15,574 adults who received RIV4 with 27,110 who received SD-IIV4, there was no statistically significant difference in the prespecified diagnoses of interest and deaths between the 2 groups. There were 35 deaths total, none of which were considered to be related to influenza vaccination.ConclusionsThis study did not identify any safety concerns regarding RIV4 use among 18–64-year-olds who self-identified as Chinese. This study supports the safety of RIV4 vaccine in this population.     

Efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens.

Fowlpox virus (FPV) recombinants expressing influenza virus H5 haemagglutinin (HA), nucleoprotein (NP) or co-expressing both of these antigens were tested for vaccine efficacy in chickens. Immunization with the recombinant FPV-HA was highly efficacious but provided no cross protection between subtypes. Bursectomy established that immunity against the H5 subtype was antibody-mediated despite the presence of very low levels of antibody in the vaccinated birds. Immunization with the recombinant FPV expressing the cross-reactive NP antigen did not provide protective immunity despite hyperimmunization and provided no benefit above HA expressed alone. The results suggest that the kinetics of viral replication outpaces immunity induced by NP.     

Enhanced cross-lineage protection induced by recombinant H9N2 avian influenza virus inactivated vaccine

Background

Antigenic drift of H9N2 low pathogenic avian influenza viruses (AIV) may result in vaccination failure in the poultry industry and thus a cross-protective vaccine against H9N2 AIV is highly desirable.

Immune responses and protection in different strains of aged mice immunized intranasally with an adjuvant-combined influenza vaccine.

Immune responses and protection against influenza virus infection were compared between young (2 months) and aged (18 months) BALB/c, C3H and C57BL/6 (B6) mice after intranasal vaccination. The mice were immunized with 2.5 microg protein of A/PR/8/34 (PR8) (H1N1) virus vaccine containing a cholera toxin adjuvant. In both the young and aged BALB/c mice, high levels of PR8-specific antibody-forming cell (AFC) responses were induced in the nasal-associated lymphoid tissue (NALT) 7 days after immunization. Nasal wash IgA and serum IgG antibody (Ab) responses to the PR8 haemagglutinin (HA) 4 weeks after immunization were slightly higher in the young mice than in the aged mice. The young mice showed complete protection against challenge infection, while the aged mice showed only a partial protection. In the C3H mice, NALT-AFC, and IgA and IgG Ab responses were higher in the young mice than those in the aged mice in parallel with the more efficient protection in the young mice than in the aged mice. Both the young and aged B6 mice showed no NALT-AFC responses, scarce IgA and IgG Ab responses and no protection. In the BALB/c mice, IgG1 and IgG2a levels were significantly lower in the aged mice. On the other hand, in the C3H mice, only IgG2a level was significantly lower in the aged mice. Similar results were obtained in terms of immune responses and protection between the young and aged mice of three different strains of mice after intra-nasal immunization with 0.1 microg of PR8 vaccine containing the adjuvant, two-times at 4-week intervals. In the B6 mice, the immune response was improved by immunization with a higher dose of the adjuvant-combined vaccine. These results suggest that local Ab responses, as well as systemic Ab responses, are downregulated in aged mice, although the degree of the downregulation of immune responses differs from strain to strain.     

Induction of protective class I MHC-restricted CTL in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant.

Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines. The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses. The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2). Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses. In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants. Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199). D protein without adjuvant did not elicit CTL, regardless of the route of injection. However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant. In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection. Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection. Passive transfer of immune sera from CB6F1 mice was also not protective. This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza.     

DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice

Influenza vaccination practice, which is based on neutralizing antibodies, requires being able to predict which viral strains will be circulating. If an unexpected strain, as in the 1997 H5N1 Hong Kong outbreak, or even a pandemic emerges, appropriate vaccines may take too long to prepare. Therefore, strategies based on conserved influenza antigens should be explored. We studied DNA vaccination in mice with plasmids expressing conserved nucleoprotein (NP) and matrix (M) from an H1N1 virus. After vaccination, mice were challenged with A/H5N1 viruses of low, intermediate, and high lethality. A/NP+A/M DNA vaccination reduced replication of A/Hong Kong/486/97 (HK/486), a nonlethal H5N1 strain, and protected against lethal challenge with more virulent A/Hong Kong/156/97 (HK/156). After HK/156 exposure, mice survived rechallenge with A/Hong Kong/483/97 (HK/483), although the DNA vaccination alone protected poorly against this highly virulent strain. In the absence of antigenically matched hemagglutinin-based vaccines, DNA vaccination with conserved influenza genes may provide a useful first line of defense against a rapidly spreading pandemic virus.     

A novel influenza subunit vaccine composed of liposome-encapsulated haemagglutinin/neuraminidase and IL-2 or GM-CSF. I. Vaccine characterization and efficacy studies in mice

The aim of this study was to improve the potency of the currently used influenza subunit vaccines, which are of relatively low efficiency in high-risk groups. Influenza A virus (Shangdong/9/93) haemagglutinin/neuraminidase (H3N2), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) were encapsulated, each separately or combined, in multilamellar vesicles composed of dimyristoyl phosphatidylcholine. BALB/c mice were immunized once, i.p. or s.c., with 0.05-2.0 microg HN administered either as free antigen (F-HN), adsorbed to aluminum hydroxide (Al-HN), or encapsulated in liposomes (Lip-HN), separately or together with 1 x 10(2)-4.5 x 10(4) units of free or encapsulated cytokines. Serum antibodies were assayed on days 11-360 by the haemagglutination-inhibition (HI) test and ELISA. Protective immunity against intranasal virus challenge was determined at 9-14 months post-vaccination. The following results were obtained: (1) The efficiency of encapsulation in liposomes was 95, 90 and 38% for HN, IL-2 and GM-CSF, respectively, and the liposomal preparations were highly stable as an aqueous dispersion for > 2 months at 4 degrees C. (2) Following immunization with 0.5 microg Lip-HN, there was an earlier, up to 50-fold stronger, and 3-5 times longer response than that obtained with nonliposomal HN. (3) Coimmunization with free cytokines further increased the response 2-20 times and the two cytokines had an additive effect. (4) Liposomal cytokines were 2-20 times more effective than the free cytokines and their stimulatory effect was more durable. (5) A 100% seroconversion (HI titer > or = 40) was achieved with only 10-25% of the routinely used antigen dose, by encapsulating either antigen or cytokine. (6) The level of protection following vaccination with the combined liposomal vaccines was 70-100% versus 0-25% in mice immunized with Al-HN alone, and no toxicity was observed. In conclusion, our animal experiments show that the liposomal vaccines are superior to the currently used influenza vaccines, increasing the response by 2-3 orders of magnitude in mice. This approach may also prove valuable for subunit vaccines against other microorganisms.