Influenza Neuraminidase Characteristics and Potential as a Vaccine Target.

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

FRETing over SARS-CoV-2: Conformational Dynamics of the Spike Glycoprotein.

In this issue of Cell Host & Microbe, Lu et al. utilize single-molecule FRET to reveal the conformation dynamics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, showing transitions from a closed ground state to the open receptor-accessible conformation via an on-path intermediate. These insights into spike conformations will facilitate rational immunogen design.

The role of pi-interactions and hydrogen bonds in fully protective synthetic malaria vaccine development.

Analysis of our Plasmodium falciparum malaria parasite peptides' 1H-NMR database in the search for H-bonds and π-interactions led us to correlate their presence or absence with a peptide's particular immunological behavior. It was concluded that a 26.5 ± 1.5 Å between positions 1 to 9 of the HLA-DRß1* interacting region was necessary for proper docking of 20mer-long peptides and these MHC Class II molecules for full-protective immunity. Presence of intramolecular H-bonds or π-interactions leading to righ-handed α-helix or ß-turn conformation in this peptide's region induces different immune responses or none. PPIIL conformation and the absence of any intramolecular interaction thus became the first feature characterising our immune protection-inducing structures as malaria vaccine candidates.

Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels.

Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

Canarypox and fowlpox viruses as recombinant vaccine vectors: an ultrastructural comparative analysis.

Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox virus (FP) represent efficient and safe vaccine vectors, as they correctly express transgenes in human cells, elicit complete immune responses, and show protective efficacy in preclinical animal models. At present, no information is available on the differences in the abortive replication of these two avipox viruses in mammalian cells. In the present study, the replicative cycles of CP and FP, wild-type and recombinants, are compared in permissive and non-permissive cells, using transmission electron microscopy. We demonstrate that in non-permissive cells, the replicative cycle is more advanced in FP than in CP, that human cells, whether immune or not, are less permissive to avipox replication than monkey cells, and that the presence of virus-like particles only occurs after FP infection. Overall, these data suggest that the use of FP recombinants is more appropriate than the use of CP for eliciting an immune response.

Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids.

The icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid-like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C-terminal basic domain by the 17 kDa Staphylococcus aureus nuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild-type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy.

Oral delivery of purified lipoprotein OspA protects mice from systemic infection with Borrelia burgdorferi.

The lipoprotein outer surface protein A (OspA) of the Lyme disease agent. Borrelia burgdorferi, has provided protection to mice and other animals against systemic infection when delivered orally as a recombinant protein in Escherichia coli, bacille Calmette. Guerin or Salmonella typhimurium. In the present study purified recombinant strain B31 OspA or outer surface protein D (OspD), another lipoprotein of B. burgdorferi, were administered either subcutaneously (s.c.) or orally without cell carrier or adjuvant to mice. In comparison to the OspD preparation, the OspA protein was 256-fold more resistant to trypsin. Whereas OspA in the suspension was in regular complexes of 17-25 nm in size, OspD formed amorphous globules of different sizes. Animals received a primary immunization and at least one booster. Mice immunized s.c. with either OspA or OspD had detectable antibodies to B. burgdorferi by enzyme-linked immunosorbent assay (ELISA), growth inhibition assay (GIA) and immunoblot. Delivered orally, OspA but not OspD elicited a specific antibody response, including IgA, as determined by these assays. The geometric mean titre of sera from mice who received 4 micrograms of OspA orally on days 1, 2, 4, 21 and 22 was 1470 by Ig ELISA, 320 by IgA ELISA and 128 by GIA. In infectious challenge experiments with B. burgdorferi strain Sh2-2-82 (OspA+ OspD- ) inoculated intradermally at 100 x the ID 50 all eight mice immunized with the 4 micrograms dose of OspA were protected, none of the mice immunized with the 4 micrograms dose of OspD were protected (P < 0.001 by Fisher exact test). These studies indicate that the lipoprotein OspA provides protection against systemic B. burgdorferi infection when delivered orally as a purified protein.

Preparation of proteosome-based vaccines. Correlation of immunogenicity with physical characteristics.

In order to facilitate the use of proteosome-based vaccines, we have identified and analyzed the parameters that affect their immunogenicity. As a model system we used synthetic peptides (LCF6) containing sequences from the immunodominant (NANP)n tandem repeat region of the P. falciparum circumsporozoite protein, hydrophobically complexed to multimeric protein preparations (proteosomes) of meningococcal outer membrane proteins (OMP), since we have previously shown that high levels of anti-(NANP)n IgG can be elicited in mice by use of this novel adjuvant system (Lowell et al., 1988a). We have now examined these preparations by velocity sedimentation and measured their ability to elicit an IgG response in mice. Velocity sedimentation of freshly mixed OMP and LCF6, without dialysis, produced a limited number of small complexes, whereas dialysis of the mixture for 4 d yielded heterogeneously sized complexes that became more homogeneous when the dialysis was carried out for 7 or 10 days. The most homogeneous of these peptide-proteosome complexes (those dialyzed for 10 days) induced substantial levels of anti-(NANP)n IgG in mice, and shorter periods of dialysis resulted in vaccines that induced proportionately lower titers. Analysis of a series of preparations with varying LCF6: OMP ratios (w/w) showed that the degree of peptide substitution of the proteosomes was inversely proportional to the rate of sedimentation of the complexes and that there exists an optimal degree of lipopeptide complexing to the proteosomes. Our results suggest that the parameters affecting the immunogenicity of the peptide-proteosome complexes are: (i) hapten density, and (ii) size of the complex. Furthermore, sedimentation analysis of peptide-proteosome immunogens may serve as a rapidly performed assay of immunogenic potency.

The iscom: an immunostimulating system.

To make purified antigens highly immunogenic, they have to be presented in several copies in the form of a microscopic or submicroscopic particle. This is the case, regardless of whether the antigens are obtained by isolation from conventional microorganisms, or from gene-manipulated cells, or synthesized. In the iscom, the antigens are attached as multimers to a 40-nm cage-like particle with a built-in adjuvant. The antigens in iscoms are rapidly transported from the injection site to the draining lymphatic organ. Iscom-borne antigens induced a 10-fold higher antibody response than the same amount of antigen in micelle form. One intranasal immunization with influenza virus iscoms induced protection to intranasal challenge infection in mice. Besides a strong antibody response in all Ig classes and isotypes, cytotoxic T cells were induced. With iscoms containing gp160 of HIV-1, cytotoxic T cells (CD8+ CD4-) were induced under restriction of class I MHC antigen. Iscoms containing the fusion protein of measles virus induced T cell clones in mice whereof one, after adoptive transfer, protected mice against intracerebral challenge infection. Protective immunity against Epstein-Barr virus (EBV)-induced tumor formation by iscoms containing gp350 of EBV has been elicited in cotton-top Tamerin monkeys. Protective immunity has also been induced against several virus infections including feline leukemia virus and against parasites, i.e., Trypanosoma cruzi, in mice.