Abstract
Herpesviruses (HVs) cause lifelong infections, and the majority of humans are infected with at least one; whether co-infection with HVs is beneficial or detrimental to the host is a current ...debate. We sought to determine if co-infection with HVs alters the magnitude or quality of the immune response to influenza infection, and, if so, the extent to which each virus affects illness outcome. We used a longitudinal study of naturally acquired influenza virus infection and linear regression modeling to determine differences in influenza viral load and shedding, cytokine levels, and severity scores, while taking into account the effects of confounding variables. Our results show co-infection with HVs does not significantly affect influenza virus kinetics or clearance, but Cytomegalovirus is associated with decreased symptom severity and increased antibody titers. Furthermore, co-infection alters cytokine levels involved in each step of the immune response. Changes in plasma and nasal wash cytokines were predominately associated with Herpes simplex virus 1/2 and Epstein-Barr virus co-infection, respectively. The cytokines most affected by HV serostatus in the plasma and nasal wash are MIP1b (adj. R2 = 0.3073) and RANTES (adj. R2 = 0.3604), respectively. The strongest effects on cytokine levels are associated with HV interactions and are primarily found in the plasma. In the nasal wash, co-infection mostly results in decreased levels of cytokines, including those implicated in immunopathology during influenza infection (e.g. IL1, MIP1, TNF-α, IL6). Understanding host-virus interactions during co-infection and how that relationship impacts disease severity can ultimately be used for better prognosis and/or prophylactic measures.
•Omicron BA.5 subvariant is now the predominant COVID-19 threat worldwide. While there are some data on pseudovirus neutralization titres to BA.4/5 in comparison to ancestral virus and BA.1 and BA.2 ...variants, live virus plaque neutralization is the definitive method to address this important question.•BA.4 and BA.5 subvariants were less susceptible to BNT162b2 or Coronavac vaccine elicited antibody neutralization than subvariants BA.1, BA.2 and BA.2.12.1. Nevertheless, three doses BNT162b2 or booster of BNT162b2 following two doses of CoronaVac elicited detectable BA.4 and BA.5 neutralizing antibody responses while those vaccinated with three doses of CoronaVac largely fail to do so.•Using live virus neutralization, we see some difference between BA.4 and BA.5 subvariants, with BA.5 being even more immune evasive than BA.4, which may help explain why BA.5 outcompeting BA.4 on the global scale.•We provide data on convalescent BA.5, BA.4 and BA.2.12.1 neutralizing titres in convalescent sera of BA.2 breakthrough infections, which reflects the immune landscape for most of the developed world at the moment, as we face the BA.5 outbreak.•BA.2 breakthrough infections in vaccinated individuals elicited higher levels of BA.4 or BA.5 neutralizing antibody compared to BA.2 infections in vaccine-naïve individuals.
BA.2.12.1, BA.4 and BA.5 subvariants of SARS-CoV-2 variant-of-concern (VOC) Omicron (B.1.1.529) are spreading globally. They demonstrate higher transmissibility and immune escape.
Determine BA.2.12.1, BA.4 and BA.5 virus plaque reduction neutralization test (PRNT) antibody titres in individuals recently vaccinated with BNT162b2 (n=20) or CoronaVac (n=20) vaccines or those convalescent from ancestral wild- type (WT) SARS-CoV-2 (n=20) or BA.2 infections with (n=17) or without (n=7) prior vaccination.
Relative to neutralization of the WT virus, those vaccinated with BNT162b2 had 4.8, 3.4, 4.6, 11.3 and 15.5-fold reductions of geometric mean antibody titres (GMT) to BA.1, BA.2, BA.2.12.1, BA.4 and BA.5 viruses, respectively. Similarly, those vaccinated with CoronaVac had 8.0, 7.0, 11.8, 12.0 and 12.0 fold GMT reductions and those with two doses of CoronaVac boosted by BNT162b2 had 6.1, 6.7, 6,3, 13.0 and 21.2 fold GMT reductions to these viruses, respectively. Vaccinated individuals with BA.2 breakthrough infections had higher GMT antibody levels vs. BA.4 (36.9) and BA.5 (36.9) than unvaccinated individuals with BA.2 infections (BA.4 GMT 8.2; BA.5 GMT 11.0).
BA.4 and BA.5 subvariants were less susceptible to BNT162b2 or CoronaVac vaccine elicited antibody neutralization than subvariants BA.1, BA.2 and BA.2.12.1. Nevertheless, three doses BNT162b2 or booster of BNT162b2 following two doses of CoronaVac elicited detectable BA.4 and BA.5 neutralizing antibody responses while those vaccinated with three doses of CoronaVac largely fail to do so. BA.2 infections in vaccinated individuals led to higher levels of BA.4 or BA.5 neutralizing antibody compared to those who were vaccine-naive.
The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 ...was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.
Abstract
Studies have identified genetic, viral, and immunological associations with the quality of anti-influenza immunity and/or influenza disease severity, however, there has not been an ...integrative analysis of these factors, both at baseline and during acute infection. Using samples from healthy or flu infected human cohorts across 5 countries, we found that herpesviruses (HVs) have unique and interactive effects on cytokine levels specific to anatomic location during flu infection. Similar cytokine associations were also observed in healthy controls. Additionally, HV infection is also associated with decreased flu severity and virus shedding, and increased antibody titers. Associations between cytokine levels and flu severity were consistent in cohorts of similar ancestral and environmental backgrounds, however unique correlates were observed in populations from distinct backgrounds. Further, we identified ~100 variants in immune related genes either enriched in or absent from a given ancestral population, a portion of which are eQTLs at baseline, supporting the potential of host genetics to impact immune variation. Ongoing work focuses on assessing which SNPs are eQTLs in response to bacterial and viral TLR agonists. Ancestry informative marker PCA values and eQTLs will be included in the statistical models to account for the collective effects of infectious, biological, and genetic factors. These results will provide insight into which factors predominantly affect a given immune measure and how this contributes to immune competence and variation across distinct populations. Understanding these interactions will have implications for other infectious diseases, autoimmunity, clinical study design, and immunotherapy.
Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, ...all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their ‘open’ counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.
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•Raltegravir weakly inhibits the PAN endonuclease of the influenza RNA polymerase.•The structure of the PAN-raltegravir complex resembles the RNA substrate complex.•Chemically modified raltegravir to optimize its similarity to the core RNA complex.•The best derivatives bind tightly to the PAN endonuclease and are potent inhibitors.•Binding is unaffected by mutations that restrict current PAN-directed therapeutics.
Influenza A/H5N8 viruses infect poultry and wild birds in many countries. In 2021, the first human A/H5N8 cases were reported.
We conducted a phase I, cohort-randomized, double-blind, controlled ...trial of inactivated influenza A/H5N8 vaccine (clade 2.3.4.4c) administered with or without adjuvant. Cohort 1 subjects received either two doses of AS03-adjuvanted vaccine containing 3.75 μg or 15 μg hemagglutinin (HA); two doses of 15 μg HA unadjuvanted vaccine; or one dose of AS03-adjuvanted vaccine (3.75 μg or 15 μg HA), followed by one dose of non-adjuvanted vaccine (same HA content). Cohort 2 subjects received two doses of MF59-adjuvanted vaccine containing 3.75 μg or 15 μg HA, or 15 μg HA of non-adjuvanted vaccine. Subjects were followed for 13 months for safety and immunogenicity.
We enrolled 386 adult subjects in good health. Solicited adverse events were generally mild and more common among subjects who received adjuvanted vaccines. Antibody responses (hemagglutination inhibition or microneutralization assays) were highest in the two-dose AS03 group, followed by the one-dose AS03 group, the MF59 groups, and the non-adjuvanted groups. Antibody levels returned to baseline 12 months after the second vaccination in all groups except the 15 μg AS03-adjuvanted group. Cross-reactive antibodies to clade 2.3.4.4b strains isolated from recent human cases were demonstrated in a subset of both 15 μg adjuvanted groups.
Two doses of influenza A/H5N8 vaccine were well-tolerated. Immunogenicity improved with receipt of two doses of adjuvanted vaccine and higher antigen content. (Funded by the National Institute of Allergy and Infectious Diseases.
Because of the pathogenicity and low incidence of avian influenza virus infections in humans, the immune correlates of protection for avian influenza vaccines cannot be determined from clinical ...studies. Here, we used the ferret model to address this for an avian influenza H5N1 vaccine. Using oil-in-water adjuvants, we generated groups of ferrets with undetectable (geometric mean titer GMT < 10), low (GMT = 28.3), or high (GMT > 761.1) hemagglutination-inhibition (HAI) titers to the A/Viet Nam/1203/2004 (H5N1) virus. Ferrets were then challenged with the wild-type virus and disease severity and immunologic parameters were studied. The severity of infection and symptom profile were inversely associated with pre-challenge HAI titers in a dose-dependent manner. A vaccinated ferret with no detectable HAI-antibodies but high flu-specific IgG-antibody titers mounted rapid functional antibodies after infection and experienced milder disease compared to other ferrets in the group. Compared to naïve ferrets, all vaccinated ferrets showed improved cellular immunity in the lungs and peripheral blood. High number of IFNγ
CD8- T cells in the airways was associated with early viral clearance. Thus, while neutralizing antibodies are the best correlate of protection, non-neutralizing antibodies can also be protective. This should be taken into consideration in future avian influenza vaccine trials.
Background. The relationship between influenza virus infectivity and virus shedding, based on different diagnostic methods, has not been defined. Methods. Three donor ferrets infected with 2009 ...pandemic influenza A(H1N1) underwent daily quantitative culture, antigen-detection testing, and real-time reverse transcription-polymerase chain reaction (RT-PCR). Eight contacts were sequentially cohoused with each of the donors for 24 hours during days 3–10 after inoculation. Results. Transmission was observed until day 5 after inoculation, corresponding to high culture titers and positive results of antigen-detection tests. Real-time RT-PCR showed no relation to the cessation of transmission. Conclusions. Antigen-detection testing and virus culture but not real-time RT-PCR identified the end of the infectious period.
Past pandemic influenza viruses with sustained human-to-human transmissibility have emerged from animal influenza viruses. Employment of experimental models to assess the pandemic risk of emerging ...zoonotic influenza viruses provides critical information supporting public health efforts. Ferret transmission experiments have been utilized to predict the human-to-human transmission potential of novel influenza viruses. However, small sample sizes and a lack of standardized protocols can introduce interlaboratory variability, complicating interpretation of transmission experimental data. To assess the range of variation in ferret transmission experiments, a global exercise was conducted by 11 laboratories using two common stock H1N1 influenza viruses with different transmission characteristics in ferrets. Parameters known to affect transmission were standardized, including the inoculation route, dose, and volume, as well as a strict 1:1 donor/contact ratio for respiratory droplet transmission. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored and analyzed. The overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. Among environmental parameters that varied across laboratories, donor-to-contact airflow directionality was associated with increased transmissibility. To attain high confidence in identifying viruses with moderate to high transmissibility or low transmissibility under a smaller number of participating laboratories, our analyses support the notion that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment. IMPORTANCE Following detection of a novel virus, rapid characterization efforts (both in vitro and in vivo) are undertaken at numerous laboratories worldwide to evaluate the relative risk posed to human health. Aggregation of these data are critical, but the use of nonstandardized protocols can make interpretation of divergent results a challenge. For evaluation of virus transmissibility, a multifactorial trait which can only be evaluated in vivo, identifying intrinsic levels of variability between groups can improve the utility of these data, as well as ensure that experiments are performed with sufficient replication to ensure high confidence in compiled results. Using the ferret transmission model and two influenza A viruses, we conducted a multicenter standardization exercise to improve the interpretation of transmission data generated during risk assessment activities; this exercise serves as a model for future efforts employing both in vitro and in vivo models against possible pandemic pathogens.
The Animal Biosafety Level 3 Enhanced (ABSL-3+) laboratory at St. Jude Children's Research Hospital has a long history of influenza pandemic preparedness. The emergence of SARS-CoV-2 and subsequent ...expansion into a pandemic has put new and unanticipated demands on laboratory operations since April 2020. Administrative changes, investigative methods requiring increased demand for inactivation and validation of sample removal, and the adoption of a new animal model into the space required all arms of our Biorisk Management System (BMS) to respond with speed and innovation.
In this report, we describe the outcomes of three major operational changes that were implemented to adapt the ABSL-3+ select agent space into a multipathogen laboratory. First were administrative controls that were revised and developed with new Institutional Biosafety Committee protocols, laboratory space segregation, training of staff, and occupational health changes for potential exposure to SARS-CoV-2 inside the laboratory. Second were extensive inactivation and validation experiments performed for both highly pathogenic avian influenza and SARS-CoV-2 to meet the demands for sample removal to a lower biosafety level. Third was the establishment of a new caging system to house Syrian Golden hamsters for SARS-CoV-2 risk assessment modeling.
The demands placed on biocontainment laboratories for response to SARS-CoV-2 has highlighted the importance of a robust BMS. In a relatively short time, the ABSL-3+ was able to adapt from a single select agent space to a multipathogen laboratory and expand our pandemic response capacity.
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