SARS-CoV-2 Omicron variant causes mild pathology in the upper and lower respiratory tract of hamsters.

Since its discovery in 2019, multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been identified. This study investigates virus spread and associated pathology in the upper and lower respiratory tracts of Syrian golden hamsters at 4 days post intranasal SARS-CoV-2 Omicron infection, in comparison to infection with variants of concern (VOCs) Gamma and Delta as well as ancestral strain 614 G. Pathological changes in the upper and lower respiratory tract of VOC Omicron infected hamsters are milder than those caused by other investigated strains. VOC Omicron infection causes a mild rhinitis with little involvement of the olfactory epithelium and minimal lesions in the lung, with frequent sparing of the alveolar compartment. Similarly, viral antigen, RNA and infectious virus titers are lower in respiratory tissues of VOC Omicron infected hamsters. These findings demonstrate that the variant has a decreased pathogenicity for the upper and lower respiratory tract of hamsters.

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays notable immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other variants of concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.

Development of molecular clamp stabilized hemagglutinin vaccines for Influenza A viruses.

Influenza viruses cause a significant number of infections and deaths annually. In addition to seasonal infections, the risk of an influenza virus pandemic emerging is extremely high owing to the large reservoir of diverse influenza viruses found in animals and the co-circulation of many influenza subtypes which can reassort into novel strains. Development of a universal influenza vaccine has proven extremely challenging. In the absence of such a vaccine, rapid response technologies provide the best potential to counter a novel influenza outbreak. Here, we demonstrate that a modular trimerization domain known as the molecular clamp allows the efficient production and purification of conformationally stabilised prefusion hemagglutinin (HA) from a diverse range of influenza A subtypes. These clamp-stabilised HA proteins provided robust protection from homologous virus challenge in mouse and ferret models and some cross protection against heterologous virus challenge. This work provides a proof-of-concept for clamp-stabilised HA vaccines as a tool for rapid response vaccine development against future influenza A virus pandemics.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Protective efficacy of a polyvalent influenza A DNA vaccine against both homologous (H1N1pdm09) and heterologous (H5N1) challenge in the ferret model.

This study describes the protective efficacy of a novel influenza plasmid DNA vaccine in the ferret challenge model. The rationally designed polyvalent influenza DNA vaccine encodes haemagglutinin and neuraminidase proteins derived from less glycosylated pandemic H1N1 (2009) and H3N2 (1968) virus strains as well as the nucleoprotein (NP) and matrix proteins (M1 and M2) from a different pandemic H1N1 (1918) strain. Needle-free intradermal immunisation with the influenza DNA vaccine protected ferrets against homologous challenge with an H1N1pdm09 virus strain, demonstrated by restriction of viral replication to the upper respiratory tract and reduced duration of viral shedding post-challenge. Breadth of protection was demonstrated in two heterologous efficacy experiments in which animals immunised with the influenza DNA vaccine were protected against challenge with a highly pathogenic avian influenza H5N1 virus strain with reproducible survival and clinical outcomes.

Animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.

Immunogenicity and efficacy comparison of MDCK cell-based and egg-based live attenuated influenza vaccines of H5 and H7 subtypes in ferrets.

During a pandemic, the availability of specific pathogen free chicken eggs is a major bottleneck for up-scaling response to the demand for influenza vaccine. This has led us to explore the use of Madin-Darby Canine Kidney (MDCK) cells for the manufacture of live attenuated influenza vaccine (LAIV) that provides production flexibility and speed. The present study reports the comparison of the immunogenicity and efficacy of two MDCK-based LAIVs against two egg-based LAIVs prepared from the same pandemic potential strains of H5 and H7 subtypes after a single dose of the vaccine followed by a challenge with a homologous wild type strain. The vaccine strains have been generated by classical method of reassortment using the A/Leningrad/134/17/57 master donor strain. Additionally, a prime-boost regimen of the MDCK-based vaccine followed by a challenge with a homologous wild type strain for H5 and H7 immunized ferrets and also a heterologous wild type strain for the H5 immunized animals was studied. No difference in the hemagglutination inhibition and virus neutralization antibody titers against the homologous virus was observed following a single dose of either egg-based or MDCK-based H5 and H7 LAIV vaccine. A second dose of MDCK-based vaccine significantly boosted antibody titers in the vaccinated animals. Both a single dose or two doses of LAIV provided complete protection from lower respiratory tract infection and resulted in a significant reduction in the virus titers recovered from the throat, nasal turbinates and lungs after challenge with the homologous wild type strain. Protection from a challenge with a heterologous strain of H5 was also observed after two doses of the MDCK-based LAIVs. This data strongly supports the use of MDCK as a substrate for the manufacture of LAIV which ensures reliable quality, safety, production flexibility, speed and breadth of protection, features that are highly critical during a pandemic.

Cross-Protective Potential and Protection-Relevant Immune Mechanisms of Whole Inactivated Influenza Virus Vaccines Are Determined by Adjuvants and Route of Immunization.

Adjuvanted whole inactivated virus (WIV) influenza vaccines show promise as broadly protective influenza vaccine candidates. Using WIV as basis we assessed the relative efficacy of different adjuvants by carrying out a head-to-head comparison of the liposome-based adjuvants CAF01 and CAF09 and the protein-based adjuvants CTA1-DD and CTA1-3M2e-DD and evaluated whether one or more of the adjuvants could induce broadly protective immunity. Mice were immunized with WIV prepared from A/Puerto Rico/8/34 (H1N1) virus intramuscularly with or without CAF01 or intranasally with or without CAF09, CTA1-DD, or CTA1-3M2e-DD, followed by challenge with homologous, heterologous or heterosubtypic virus. In general, intranasal immunizations were significantly more effective than intramuscular immunizations in inducing virus-specific serum-IgG, mucosal-IgA, and splenic IFNγ-producing CD4 T cells. Intranasal immunizations with adjuvanted vaccines afforded strong cross-protection with milder clinical symptoms and better control of virus load in lungs. Mechanistic studies indicated that non-neutralizing IgG antibodies and CD4 T cells were responsible for the improved cross-protection while IgA antibodies were dispensable. The role of CD4 T cells was particularly pronounced for CTA1-3M2e-DD adjuvanted vaccine as evidenced by CD4 T cell-dependent reduction of lung virus titers and clinical symptoms. Thus, intranasally administered WIV in combination with effective mucosal adjuvants appears to be a promising broadly protective influenza vaccine candidate.

Cross-Protective Immune Responses Induced by Sequential Influenza Virus Infection and by Sequential Vaccination With Inactivated Influenza Vaccines.

Sequential infection with antigenically distinct influenza viruses induces cross-protective immune responses against heterologous virus strains in animal models. Here we investigated whether sequential immunization with antigenically distinct influenza vaccines can also provide cross-protection. To this end, we compared immune responses and protective potential against challenge with A(H1N1)pdm09 in mice infected sequentially with seasonal A(H1N1) virus followed by A(H3N2) virus or immunized sequentially with whole inactivated virus (WIV) or subunit (SU) vaccine derived from these viruses. Sequential infection provided solid cross-protection against A(H1N1)pdm09 infection while sequential vaccination with WIV, though not capable of preventing weight loss upon infection completely, protected the mice from reaching the humane endpoint. In contrast, sequential SU vaccination did not prevent rapid and extensive weight loss. Protection correlated with levels of cross-reactive but non-neutralizing antibodies of the IgG2a subclass, general increase of memory T cells and induction of influenza-specific CD4+ and CD8+ T cells. Adoptive serum transfer experiments revealed that despite lacking neutralizing activity, serum antibodies induced by sequential infection protected mice from weight loss and vigorous virus growth in the lungs upon A(H1N1)pdm09 virus challenge. Antibodies induced by WIV vaccination alleviated symptoms but could not control virus growth in the lung. Depletion of T cells prior to challenge revealed that CD8+ T cells, but not CD4+ T cells, contributed to cross-protection. These results imply that sequential immunization with WIV but not SU derived from antigenically distinct viruses could alleviate the severity of infection caused by a pandemic and may improve protection to unpredictable seasonal infection.

Comparison of single radial immunodiffusion, SDS-PAGE and HPLC potency assays for inactivated influenza vaccines shows differences in ability to predict immunogenicity of haemagglutinin antigen.

The current gold-standard potency test for inactivated influenza vaccines is the single radial immunodiffusion (SRD) assay. A number of alternative potency tests for inactivated influenza vaccines have been proposed in recent years. Evaluation of these new potency tests commonly involves comparison with SRD, in order to ascertain that the new method obtains values that correlate with those measured by the standard potency test. Here, we extended comparison of two methods, reverse-phase HPLC and SDS-PAGE, with SRD by assessing the methods' capacity to detect loss of potency induced by various deliberate treatments of vaccine samples. We demonstrate that neither of these methods detected the loss of potency observed by SRD; importantly, neither SDS-PAGE nor reverse-phase HPLC reflected results from mouse experiments that showed decreased immunogenicity and protection in vivo. These results emphasise the importance of assessing the stability-indicating nature, ie the ability to measure loss of vaccine potency, of any potential new potency assay.

Matrix M H5N1 Vaccine Induces Cross-H5 Clade Humoral Immune Responses in a Randomized Clinical Trial and Provides Protection from Highly Pathogenic Influenza Challenge in Ferrets.

BACKGROUND AND METHODS: Highly pathogenic avian influenza (HPAI) viruses constitute a pandemic threat and the development of effective vaccines is a global priority. Sixty adults were recruited into a randomized clinical trial and were intramuscularly immunized with two virosomal vaccine H5N1 (NIBRG-14) doses (21 days apart) of 30 µg HA alone or 1.5, 7.5 or 30 µg HA adjuvanted with Matrix M. The kinetics and longevity of the serological responses against NIBRG-14 were determined by haemagglutination inhibition (HI), single radial haemolysis (SRH), microneutralization (MN) and ELISA assays. The cross-H5 clade responses in sera were determined by HI and the antibody-secreting (ASC) cell ELISPOT assays. The protective efficacy of the vaccine against homologous HPAI challenge was evaluated in ferrets. RESULTS: The serological responses against the homologous and cross-reactive strains generally peaked one week after the second dose, and formulation with Matrix M augmented the responses. The NIBRG-14-specific seroprotection rates fell significantly by six months and were low against cross-reactive strains although the adjuvant appeared to prolong the longevity of the protective responses in some subjects. By 12 months post-vaccination, nearly all vaccinees had NIBRG-14-specific antibody titres below the protective thresholds. The Matrix M adjuvant was shown to greatly improve ASC and serum IgG responses following vaccination. In a HPAI ferret challenge model, the vaccine protected the animals from febrile responses, severe weight loss and local and systemic spread of the virus. CONCLUSION: Our findings show that the Matrix M-adjuvanted virosomal H5N1 vaccine is a promising pre-pandemic vaccine candidate. TRIAL REGISTRATION: ClinicalTrials.gov NCT00868218.

Intranasal vaccination with a plant-derived H5 HA vaccine protects mice and ferrets against highly pathogenic avian influenza virus challenge.

Highly pathogenic avian influenza H5N1 infection remains a public health threat and vaccination is the best measure of limiting the impact of a potential pandemic. Mucosal vaccines have the advantage of eliciting immune responses at the site of viral entry, thereby preventing infection as well as further viral transmission. In this study, we assessed the protective efficacy of hemagglutinin (HA) from the A/Indonesia/05/05 (H5N1) strain of influenza virus that was produced by transient expression in plants. The plant-derived vaccine, in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP) was used for intranasal immunization of mice and ferrets, before challenge with a lethal dose of the A/Indonesia/05/05 (H5N1) virus. Mice vaccinated with 15 µg or 5 µg of adjuvanted HA survived the viral challenge, while all control mice died within 10 d of challenge. Vaccinated animals elicited serum hemagglutination inhibition, IgG and IgA antibody titers. In the ferret challenge study, all animals vaccinated with the adjuvanted plant vaccine survived the lethal viral challenge, while 50% of the control animals died. In both the mouse and ferret models, the vaccinated animals were better protected from weight loss and body temperature changes associated with H5N1 infection compared with the non-vaccinated controls. Furthermore, the systemic spread of the virus was lower in the vaccinated animals compared with the controls. Results presented here suggest that the plant-produced HA-based influenza vaccine adjuvanted with c-di-GMP is a promising vaccine/adjuvant combination for the development of new mucosal influenza vaccines.

An additional oligosaccharide moiety in the HA of a pandemic influenza H1N1 candidate vaccine virus confers increased antigen yield in eggs.

The H1N1 influenza pandemic in 2009 highlighted the need for the rapid generation of candidate vaccine viruses (CVVs) against an A/California/7/2009-like virus. The first available CVVs gave low protein yields in eggs but improved yields were achieved for second generation CVVs which contained amino acid substitutions compared to their precursor viruses. In this study, we investigated the basis for the increased virus protein yield of CVV NIBRG-121xp and whether the improved yield characteristics could be transferred between this virus and two other CVVs, NYMC X-179A and NYMC X-181. We generated variant viruses by reverse genetics to contain combinations of amino acid substitutions found in high yielding NIBRG-121xp and NYMC X-181. We found that the increase in total protein yield and functional HA yield of NIBRG-121xp in eggs is attributable to the single amino acid substitution K119N in the HA. We also found that the glycosylation of position 119 is essential for the improved virus protein yield in eggs. However, the K119N yield-enhancing effect was not transferable between viruses, nor was the N129D change found in high yielding NYMC X-181. However, position 119 may be a useful locus to monitor in future for viruses and CVVs with potentially high yield.

Improved antigen yield in pandemic H1N1 (2009) candidate vaccine viruses with chimeric hemagglutinin molecules.

The candidate pandemic H1N1 vaccine virus NIBRG-121 was derived by reverse genetics and comprises the hemagglutinin (HA) and neuraminidase (NA) genes from A/California/7/2009 (CAL) on an A/Puerto Rico/8/34 (PR8) backbone. NIBRG-121 was found to grow poorly in eggs, compared to seasonal H1N1 candidate vaccine viruses. Based on our previous study with H5N1 candidate vaccine viruses, we generated two new viruses with chimeric PR8/CAL HA genes. Here we show that these new viruses have considerably improved growth in eggs and are therefore better candidate vaccine viruses for use in production of pandemic H1N1 (2009) vaccine.

The development of vaccine viruses against pandemic A(H1N1) influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

Improved haemagglutinin antigen content in H5N1 candidate vaccine viruses with chimeric haemagglutinin molecules.

The candidate vaccine virus NIBRG-14 was derived by reverse genetics and comprises the haemagglutinin (HA) and neuraminidase (NA) genes derived from the clade 1 virus A/Viet Nam/1194/2004 on an A/Puerto Rico/8/34 (PR8) backbone. The HA gene was modified to remove the multibasic cleavage site motif associated with high pathogenicity. Reports from manufacturers, confirmed by data generated in this laboratory, have shown that this virus yields a low amount of HA antigen. We have generated a panel of new viruses using reverse genetics in which each virus consists of the PR8 backbone, the NA gene from A/Viet Nam/1194/2004 and a chimeric HA gene with sequences from both PR8 and A/Viet Nam/1194/2004. Here we show that a number of these viruses have improved HA antigen content and yield and are therefore better candidate vaccine viruses for use in production of H5N1 vaccine.