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Influenza virus causes acute upper and lower respiratory infections and is the most likely, among known pathogens, to cause a large epidemic in humans. Influenza virus mutates rapidly, enabling it to evade natural and vaccine-induced immunity. Furthermore, influenza viruses can cross from animals to humans, generating novel, potentially pandemic strains. Currently available influenza vaccines induce a strain specific response and may be ineffective against new influenza viruses. The difficulty in predicting circulating strains has frequently resulted in mismatch between the annual vaccine and circulating viruses. Low-resource countries remain mostly unprotected against seasonal influenza and are particularly vulnerable to future pandemics, in part, because investments in vaccine manufacturing and stockpiling are concentrated in high-resource countries. Antibodies that target conserved sites in the hemagglutinin stalk have been isolated from humans and shown to confer protection in animal models, suggesting that broadly protective immunity may be possible. Several innovative influenza vaccine candidates are currently in preclinical or early clinical development. New technologies include adjuvants, synthetic peptides, virus-like particles (VLPs), DNA vectors, messenger RNA, viral vectors, and attenuated or inactivated influenza viruses. Other approaches target the conserved exposed epitope of the surface exposed membrane matrix protein M2e. Well-conserved influenza proteins, such as nucleoprotein and matrix protein, are mainly targeted for developing strong cross-protective T cell responses. With multiple vaccine candidates moving along the testing and development pipeline, the field is steadily moving toward a product that is more potent, durable, and broadly protective than previously licensed vaccines., (Copyright © 2016 World Health Organization. Published by Elsevier Ltd.. All rights reserved.)

Factor H binding protein (fHbp), one of the main antigens of new vaccines against serogroup B meningococcus, varies in amino acid sequence and level of expression in different clinical isolates. To evaluate the contribution of amino acid sequence variability to vaccine coverage, we constructed a strain that is susceptible to bactericidal killing only by anti-fHbp antibodies and engineered it to express equal levels of 10 different fHbp sub-variants from a constitutive promoter. Testing of these isogenic strains showed that sera from mice or adult volunteers vaccinated with fHbp variant 1.1 were bactericidal against all sub-variants 1 sequences, however the titer against the most distant sequences were several times lower. Sera from vaccinated infants were more susceptible to amino acid variations and they had lower or no bactericidal activity against the distant sub-variants 1 sequences in comparison with sera from adults given the same vaccines. The low coverage provided by fHbp could be overcome using a multicomponent vaccine. We conclude that fHbp is a very important antigen that induces bactericidal antibodies in animals, adults and infants. However, given its high variability of sequence and expression level, it is unlikely that fHbp alone can provide good protection in infants against the distant amino acid sequence variants and therefore multicomponent vaccines inducing protective immunity also against other antigens are more likely to induce a broad protective immunity in all age groups., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8(+) T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press) and support a subtype C in-frame gagpol fusion gene vaccine.

A pneumococcal conjugate vaccine (PCV) has been developed consisting of capsular polysaccharide (Ps) coupled to the outer membrane protein complex of Neiserria meningitidis serogroup B. Experiments were conducted in infant rhesus monkeys to assess the potential to administer multiple Pn types in a single vaccine. A single type conjugate, 6B, was dosed from 0.025 to 25 microg Ps. Peak anti-6B Ps Ab titers were seen at lower doses of 0.025 and 0.25 microg Ps, while reduced titers of anti-6B Ps Ab were observed at the highest doses of conjugate administered, 2.5 and 25 microg Ps. By mixing free Ps, carrier, or another monovalent PCV with this 6B PCV, it was determined that reduced anti-6B Ps titers at high PCV doses were associated only with the quantity of type-specific Ps in the conjugate. Thus, increasing the amount of carrier protein or adding an additional monovalent conjugate did not significantly affect the response to type 6B Ps. These results suggest that, given an appropriately determined dose per individual pneumococcal Ps type, a multivalent PCV that includes many different types should have satisfactory clinical immunogenicity.

The immunogenicity and protective efficacy of DNA vaccines have been amply demonstrated in numerous animal models of infectious disease. However, the feasibility of DNA vaccines for human use is not yet known. In order to investigate potential means of increasing the potency of DNA vaccines, conventional adjuvants such as aluminum salts were tested. Coadministration of these adjuvants with DNA vaccines substantially enhanced the ability of these vaccines to induce antibody responses up to 100-fold in mice and guinea pigs, and 5-10-fold in non-human primates. Effective formulations had no demonstrable effect on the levels of antigen expression in situ and consisted of adjuvants that did not form complexes with the plasmid DNA; rather they exerted their effects on antigen after expression in situ. Therefore, the potency of DNA vaccines both in laboratory rodents and in non-human primates can be substantially increased by simple formulation with conventional aluminum adjuvants.

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.

Influenza is a leading cause of morbidity and mortality in older persons. The current influenza vaccine is only modestly successful, in part because of an age-related decline in immunogenicity and also because it induces only type-specified immunity. To overcome this, we evaluated DNA vaccines encoding A/PR8/34 haemagglutinin (HA) and nucleoprotein (NP) in young and aged BALB/c mice. Control mice were given formalin-inactivated A/PR8/34, control DNA, or a non-lethal dose of PR8. Aged mice given HA DNA developed slightly lower anti-HA serum antibodies than young mice; however, both young and aged mice were protected from a homotypic PR8 challenge. Following vaccination with NP DNA, both young and aged mice developed anti-NP bulk cytotoxic T-lymphocyte (CTL) activity and pCTL frequency similar to control animals. When challenged with a low dose of A/HK/68 (H3N2) influenza virus, both young mice and aged mice showed significant protection as measured by inhibition of weight loss. When challenged with a relatively high dose of A/HR/68 (H3N2) influenza virus, however, the anti-NP vaccine only partially protected young mice and failed to protect aged mice. These data demonstrate that DNA-based vaccines are immunogenic in aged animals, but suggest that factors other than the age-related decline in CTL activity also contribute to the increased morbidity and mortality of influenza in the elderly.

Previously we showed that immunization of ferrets with DNA encoding the hemagglutinin (HA), nucleoprotein (NP), and matrix protein (M1) of influenza virus induced protective immune responses. A DNA vaccine encoding HA (from a 1991 strain), NP and M1 (from a 1989 strain) protected ferrets better against challenge with the antigenic drift variant A/Georgia/03/93 than did the inactivated vaccine from the 1992-93 influenza season. Here we report that the same DNA vaccine protected ferrets against a second, further divergent, drift variant (A/Johannesburg/33/94). Furthermore, the extent of protection provided by the DNA vaccine was equivalent to the homologous protection provided by an inactivated vaccine that exactly matched the challenge strain.

Intramuscular injection of plasmid DNA expression vectors results in transfection of myocytes in situ. To determine whether expression of antigen by myocytes is sufficient to induce protective cell-mediated immunity, stably transfected myoblasts expressing influenza nucleoprotein (NP) were transplanted into mice. These animals produced high-titer anti-NP antibodies and MHC class I-restricted cytotoxic T lymphocytes, and were protected from a cross-strain lethal challenge with influenza A virus. Therefore, antigen expression by muscle cells in vivo is sufficient to confer protective cell-mediated immunity.

We have examined in detail the characteristics of the humoral immune response and protective efficacy induced by an influenza hemagglutinin (HA) DNA vaccine. In mice injected intramuscularly with HA DNA, the magnitude of the immune responses generated, as measured by ELISA and hemagglutination inhibiting (HI) antibodies, was directly related to the amount of DNA injected and the number of doses administered. The level of anti-HA antibodies in DNA-vaccinated mice was higher than that in convalescent immune mice and was maintained for at least 1.5 years. The immunoglobulin isotype profile of the antibodies was predominantly IgG2a, similar to that induced by live virus infection but in contrast to the relative abundance of IgG1 antibodies observed after inoculation with formalin-inactivated whole virus. The presence of pre-challenge HI antibodies was found to be a good correlate of protection, in that every animal with a detectable HI titer was protected from a lethal challenge. Complete protection from a lethal dose of influenza virus (A/PR/34), as judged by 100% survival and no weight loss, was conferred by as little as 1 microgram of DNA (given twice). Furthermore, mice injected with 10 to 100 micrograms doses, when subsequently challenged with virus, showed no increase in HI titer and no production of antibodies directed against the challenge virus, suggesting a substantial inhibition of virus replication after challenge.

Papillomaviruses infect epithelia of the skin and mucous membranes and cause benign or malignant tumours in animals and in humans. The viruses are highly species-specific, and cell culture systems for propagating human papillomaviruses (HPVs) do not exist. However, there are several animal papillomavirus models. In the cottontail rabbit papillomavirus (CRPV) system, we demonstrated that recombinant CRPV virus-like particles (VLPs) consisting of the capsid proteins L1 or L1+L2 can be produced in the yeast Saccharomyces cerevisiae. Three immunizations with L1 VLPs formulated on aluminum adjuvant at 1-100 micrograms dose-1 efficiently protected rabbits from challenge with CRPV. Sera of immunized rabbits were shown to contain high-titered serum antibodies to CRPV L1 VLPs and to neutralize CRPV in vitro. Our results suggest that recombinant yeast-derived VLPs could be the basis for a candidate HPV vaccine.

Dose-response relationships were investigated between dose of influenza virus haemagglutinin (HA) or nucleoprotein (NP) DNA vaccines, and immunogenicity and protective efficacy based on humoral and cellular immunity. In mice, intramuscular (i.m.) injection of HA or NP DNA, at doses of 100 ng to 1 microgram, was found to generate haemagglutination inhibiting (HI) antibodies and cytotoxic T-lymphocytes, respectively, and provide protection in influenza virus challenge models. A direct correlation between the amount of DNA injected and the level of HI antibody was observed. In non-human primates, high-titre neutralizing antibodies were induced in animals vaccinated with as little as 10 micrograms of HA DNA. These results indicate that low doses of DNA administered by i.m. injection provide protective efficacy against influenza.