Jamieson, P. J. | bioRxiv Science

bioRxiv2021

Broadly Reactive H2 Hemagglutinin Vaccines Elicit Cross-Reactive Antibodies in Ferrets Pre-Immune to Seasonal Influenza A Viruses

Influenza vaccines have traditionally been tested in naive mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this manuscript, previously described H2 computationally optimized broadly reactive antigen (COBRA) HA vaccines (Z1, Z5) were tested in influenza virus pre-immune ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish pre-immunity. These pre-immune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or WT H2 HA recombinant proteins in a prime-boost regimen. A set of naive pre-immune or non pre-immune ferrets were also vaccinated to control of the effects of the multiple different pre-immunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with pre-existing immune responses influenced recombinant H2 HA elicited antibodies following vaccination as measured by HAI and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock vaccinated ferrets in the group. While the vast majority of the pre-immune ferrets were protected from both morbidity and mortality across all of the different pre-immunities, the Z1 COBRA HA vaccinated ferrets had significantly higher antibody titers and recognized the highest number H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines. ImportanceH1N1 and H3N2 influenza viruses have co-circulated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naive to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have pre-existing immune responses to seasonal influenza viruses. In this study, pre-immune ferrets were vaccinated with WT and COBRA H2 recombinant HA proteins in order to examine the effects of pre-existing immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination.

immunology↗

bioRxiv2020

Chemical Stabilization of the HIV-1 Capsid Results in Efficient HIV-1 Reverse Transcription in vitro

A defining activity of retroviruses is reverse transcription, the process during which the viral genomic RNA is converted into the double strand DNA required for virus replication. Reverse transcriptase (RT), the viral enzyme responsible for this process, was identified in 1970 by assaying permeabilized retrovirus particles for DNA synthesis in vitro. Such reactions are inefficient with only a small fraction of viral genomes being converted to full-length double strand DNA molecules, possibly owing to disruption of the structure of the viral core. Here we show that reverse transcription in purified HIV-1 cores is enhanced by the addition of the capsid-binding host cell metabolite inositol hexakisphosphate (IP6). IP6 potently enhanced full-length minus strand synthesis, as did hexacarboxybenzene (HCB) which also stabilizes the HIV-1 capsid. Both IP6 and HCB stabilized the association of the viral CA and RT proteins with HIV-1 cores. In contrast to the wild type, cores isolated from mutant HIV-1 particles containing intrinsically hyperstable capsids exhibited efficient reverse transcription in the absence of IP6, further indicating that the compound promotes reverse transcription by stabilizing the viral capsid. Our results show that stabilization of the HIV-1 capsid permits efficient reverse transcription in HIV-1 cores, providing a sensitive experimental system for analyzing the functions of viral and host cell molecules and the role of capsid disassembly (uncoating) in the process. IMPORTANCEHIV-1 infection requires reverse transcription of the viral genome. While much is known about the biochemistry of reverse transcription from simplified biochemical reactions, reverse transcription during infection takes place within a viral core. However, endogenous reverse transcription reactions using permeabilized virions or purified viral cores have been inefficient. Using viral cores purified from infectious HIV-1 particles, we show that efficient reverse transcription is achieved in vitro by addition of the capsid-stabilizing metabolite inositol hexakisphosphate. Enhancement of reverse transcription was linked to the capsid-stabilizing effect of the compound, consistent with the known requirement for an intact or semi-intact viral capsid for HIV-1 infection. Our results establish a biologically relevant system for dissecting the function of the viral capsid and its disassembly during reverse transcription. The system may also prove useful for mechanistic studies of emerging capsid-targeting antiviral drugs.

microbiology↗