Assessing the Reliability of SARS-CoV-2 Neutralization Studies That Use Post-Vaccination Sera

Assessing COVID-19 vaccine effectiveness against emerging SARS-CoV-2 variants is crucial for determining future vaccination strategies and other public health strategies. When clinical effectiveness data are unavailable, a common method of assessing vaccine performance is to utilize neutralization assays using post-vaccination sera. Neutralization studies are typically performed across a wide array of settings, populations and vaccination strategies, and using different methodologies. For any comparison and meta-analysis to be meaningful, the design and methodology of the studies used must at minimum address aspects that confer a certain degree of reliability and comparability. We identified and characterized three important categories in which studies differ (cohort details, assay details and data reporting details) and that can affect the overall reliability and/or usefulness of neutralization assay results. We define reliability as a measure of methodological accuracy, proper study setting concerning subjects, samples and viruses, and reporting quality. Each category comprises a set of several relevant key parameters. To each parameter, we assigned a possible impact (ranging from low to high) on overall study reliability depending on its potential to influence the results. We then developed a reliability assessment tool that assesses the aggregate reliability of a study across all parameters. The reliability assessment tool provides explicit selection criteria for inclusion of comparable studies in meta-analyses of neutralization activity of SARS-CoV-2 variants in post-vaccination sera and can also both guide the design of future neutralization studies and serve as a checklist for including important details on key parameters in publications

Three doses of COVID-19 mRNA vaccine induce class-switched antibody responses in inflammatory arthritis patients on immunomodulatory therapies

Patients with inflammatory arthritis (IA) are at increased risk of severe COVID-19 due to medication-induced immunosuppression that impairs host defenses. The aim of this study was to assess antibody and B cell responses to COVID-19 mRNA vaccination in IA patients receiving immunomodulatory therapies. Adults with IA were enrolled through the Johns Hopkins Arthritis Center and compared with healthy controls (HC). Paired plasma and peripheral blood mononuclear cell (PBMC) samples were collected prior to and 30 days or 6 months following the first two doses of mRNA vaccines (D2; HC=77 and IA=31 patients), or 30 days following a third dose of mRNA vaccines (D3; HC=11 and IA=96 patients). Neutralizing antibody titers, total binding antibody titers, and B cell responses to vaccine and Omicron variants were analyzed. Anti-Spike (S) IgG and S-specific B cells developed appropriately in most IA patients following D3, with reduced responses to Omicron variants, and negligible effects of medication type or drug withholding. Neutralizing antibody responses were lower compared to healthy controls after both D2 and D3, with a small number of individuals demonstrating persistently undetectable neutralizing antibody levels. Most IA patients respond as well to mRNA COVID-19 vaccines as immunocompetent individuals by the third dose, with no evidence of improved responses following medication withholding. These data suggest that IA-associated immune impairment may not hinder immunity to COVID-19 mRNA vaccines in most individuals

On the role of vaccine dose and antigenic distance in the transmission dynamics of Highly Pathogenic Avian Influenza (HPAI) H5N1 virus and its selected mutants in vaccinated animals

Influenza virus infections can cause high morbidity and mortality rates among animals and humans, and result in staggering direct and indirect financial losses amounting to billions of US dollars. Ever since it emerged in 1996 in Guangdong province, People’s Republic of China, one particular highly pathogenic avian influenza (HPAI) H5N1 virus has spread globally, and is responsible for massive losses of poultry, as well as human infections. For these reasons, HPAI H5N1 is considered as one of the viruses possible to cause a future influenza pandemic. One of the main reasons why influenza is a recurring problem is its ability to constantly evolve through the selection of mutants that are able to avoid immunity (be it natural or acquired). Due to the accumulation of mutations during genome replication, diverse/variant influenza genome sequences co-exist in a virus pool (quasispecies). These sequences can contain mutations that are able to confer selective advantages to the influenza virus given the opportunity. As a consequence, whenever a situation arises that places the virus under any type of pressure that the dominant virus sequence cannot cope with (i.e. immune pressure, selective receptor binding, etc.), the virus with the genome sequence that allows it to better adapt to that particular pressure becomes selected and takes over. Because of the influenza virus’s high rate of mutations, a global surveillance network is in place to monitor changes in circulating strains among humans that would warrant an update of the vaccines used. For human influenza strains, vaccines are updated frequently (every one or two years) and a similar situation holds true for racehorse vaccination. For avian influenza vaccination, however, the situation is different. In most countries, vaccination against avian influenza is not used, and in the countries where vaccines are used (either as routine or emergency measures), they are not updated as frequently as human vaccines are. In addition, in many instances vaccination against avian influenza viruses has met with some spectacular failures, since it failed to produce a level of immunity that would protect against circulating field strains. These vaccination failures have often been attributed to the fact that without constant vaccine updating (as is done for human influenza), the vaccines used are not able to keep up with continuously evolving antigenic variants selected in the field, and thus to protect poultry against them. In addition, since it is known that immune pressure resulting from vaccination can be a driving force in the evolution of influenza viruses and the selection of immune-escape mutants, there is a school of thought that posits that vaccination against avian influenza is not only a very expensive affair (especially if vaccines need to be frequently updated), but can also lead to selection of mutants that are able to avoid vaccination-induced immunity. The research reported in this thesis started with addressing the gaps in the knowledge regarding the role of vaccination-induced immunity in the selection of immune-escape mutants of HPAI H5N1, and if there is a way for vaccines to still be able to protect against antigenically-distant variants of the vaccine seed strain, without the need for frequent vaccine updates. Our first step in studying influenza virus evolution and selection of immune-escape mutants was to investigate how antigenic pressure may drive the selection of such mutants, and what the effect of the selected mutations on the pathogenicity and transmissibility of the mutants may be. Although there exist a variety of methods to select for influenza virus mutations (i.e. monoclonal antibodies, site-directed mutagenesis, reverse genetics, etc.), none of them is representative of selection as it happens in a vaccinated animal. In Chapter 2, we discuss in detail a laboratory-based system we have developed, in which immune-escape mutants are selected using homologous polyclonal chicken sera, similar to how they are selected in the field due to vaccination- induced immune pressure. We find that selection takes place early on, and additional mutations are selected when immune pressure is increased. Antigenic distances between the selected mutants and their parent strains are also increased throughout the selection process, but not in a linear fashion. Our selection system proved to be robust and replicable, and to be representative of selection in the field, since the mutations we selected for are also found in naturally-selected field isolates, and the antigenic distances between our selected mutants and their parent strains are similar to antigenic distances between vaccine strains and field isolates. We continued our research by addressing the roles played by vaccine dose (and resulting immunity) and antigenic distance between vaccine and challenge strains, in the transmission of HPAI H5N1 viruses, by employing transmission experiments using vaccinated chickens (Chapter 3). To our surprise, we found that the effect of antigenic distances between vaccine and challenge strains on transmission is very small compared to the effect of vaccine dose. We then quantified, for the first time, the minimum level of immunity and minimum percentage of the vaccinated population exhibiting said immunity, in order for vaccines to be able to protect against transmission even of strains that are antigenically distant to the vaccine seed strain. Transmission of such strains in well-vaccinated populations would allow for a scenario where vaccination- induced immunity may drive the selection of immune-escape mutants. Our results show that in order for vaccines to prevent transmission of antigenically distant strains (such as the ones resulting from selection due to immune pressure), the threshold level of immunity against these strains should be ≥23 haemagglutination inhibition units (HIU), in at least 86.5% of the vaccinated population. This level of immunity can be estimated by knowing the antigenic distance between the vaccine and challenge (field) strain, and the HI titre against the vaccine strain, which would then allow the approximate level of immunity against the field strain to be deduced. For example, assuming the HI titre against a vaccine strain is 210 HIU, and the distance with the challenge (field) strain is 24 HIU, according to our results the vaccine should be able to protect against the challenge strain, because the difference in HI titres should be around 26 HIU (i.e. above 23 HIU). These results, taken together with our previous work on selection of mutants, where we showed that the antigenic distances between our mutants and their parent strains are representative of distances found in the field, point to the fact that it is unlikely that vaccination-induced immunity can lead to selection of mutants able to escape it, given that a threshold level of immunity in a minimum percentage of the vaccinated population is achieved. As a consequence, we believe that constant vaccine updating may not be necessary for avian influenza viruses, as long as a threshold level of immunity is maintained. This makes vaccination a more attractive control measure, both from a health perspective and a financial one, than just applying biosecurity measures. To examine the effect the mutations in the haemagglutinin protein of our selected mutants may have in their transmission among chickens vaccinated with the parent strain, we used reverse genetics techniques to insert the HA gene of our most antigenically distant mutant into the parent strain backbone (Chapter 4). We vaccinated animals with a sub-optimal dose of vaccine, and we concluded that the mutations we selected for did not allow the mutant to avoid even low levels of immunity, such as the ones resulting from a sub-optimal vaccine dose (which resembles a poor field vaccination scenario). At the same time, the HA mutations we selected for did not appear to have a negative effect either on the pathogenicity of the mutant, or its ability to transmit to unvaccinated animals, since both parameters were comparable to the parent strain. Finally, we studied the role inter-animal variation in immunity – as measured by HI titres – has in the accuracy of antigenic cartography calculations (Chapter 5). We found that using sera from more than one animal significantly increased the accuracy of antigenic distance calculations, since it takes into account individual differences in immune responses to vaccination, an inevitable phenomenon documented in both humans and animals. In addition, we increased the accuracy of antigenic maps by avoiding the use of dimension-reducing algorithms as is currently done. By not reducing the dimensionality of virus positioning in space, our maps retain the original geometry between strains or sera, leading to more accurate positioning (Chapters 2 and 5). We hope that improving the accuracy of antigenic cartography can lead to a more precise surveillance of influenza evolution and better informed decisions regarding the need to update vaccines. Taken collectively, our results can improve field vaccination outcomes, since they provide guidelines on how to increase vaccination efficiency in stopping transmission of even antigenically-distant strains. In addition, our method for selecting for immune- escape mutants can be a valuable addition to research on influenza virus evolution. Moreover, policy making decisions regarding vaccination against any type of influenza can also benefit from our improvement on antigenic cartography accuracy, saving unnecessary costs in vaccine updating, and reducing morbidity and mortality of both animals and humans.</p

Physicochemical study of aerosols in the atmosphere of attica basin

The scope of the present Thesis was the chemical characterization of atmospheric aerosol in the atmosphere of Athens basin according to semivolatile organic compounds, SOCs and the study of its physicochemical properties related to the gas/particle partition of SOCs in the atmosphere.In the present Thesis experimental part is consisted of two parts: 1. Study of SOCs concentration in the atmosphere of Attica basin Data concerning particulate and gas phase concentrations of SOCs (PAHs, n-alkanes and PCBs) in the atmosphere of Athens basin are presented in this study for a 17 month period sampling in three sites: an urban heavy traffic site, a semi-urban site influenced by heavy duty diesel vehicles and an industrialized site located in West Attica landfill, influenced by an accidental lanfill fire. It was shown that: • Levels of SOCs were found to be similar to other sites of the same nature in Europe, #sia, North and South America while a weak reducing tendency was found for PAHs levels when comparing to data for a similar study in Patission street, during 1984-1986 • Vehicle traffic emissions were found to be the major source of PAHs and n-alkanes. • PCBs were originated probably by air-surface exchange. • Other sources of SOCs less important, are biogenic for n-alkanes, and industrial activities situated in the south-west region of Attica basin for PAHs and PCBs. • Biogenic sources of n-alkanes become more important during autumn. • Meso-scale air masses circulation induced by Attica basin topography and meteorology, was found to influence significantly atmospheric levels of SOCs. South winds favorite SOCs accumulation and along with west wind are responsible for transport of SOCs from industrialized areas. Data also provided evidence of SOCs both ways transport between Attica basin and Thriasio Plain. • Lanfill fire incident resulted to elevated levels in the atmosphere for selected PAHs and PCBs. 2. Study of gas/particle partition of SOCs in the atmosphere of Attica basin Temperature dependence of partition showed that: • Temperature was found to be not the only factor controlling gas phase concentration and partition constant of SOCs, giving evidence for local emissions impact and transport from adjacent areas. • Meso-scale air masses circulation was also found, for some SOCs, to provide conditions for discernible types of aerosol according to physical and chemical properties of their partition. Both descriptions, using either vapor pressure or octanol-air partition coefficient were applied in order to parameterize SOCs partition in Athens atmosphere. It was shown that: • Deviations from the prediction models were found in both cases and were attributed to nonexchangeable fraction of pyrogenic volatile SOCs. • Non-equilibrium state probably results by proximity to the sources, slow sorption or desorption of non-volatile SOCs due to low diffusion rates and atmospheric reaction of SOCs in gas and particle phase. The octanol-air model was used to calculate theoretical partition constants. The octanol,soot-air model was applied as well, to calculate theoretical partition constants for each sampling event It was found that: • Octanol-air, soot-air combined model was found to predict accurately PAHs partition especially when air masses were originated by heavy emission areas. • Octanol-air, soot-air combined model also gave PCBs partition constants closer to the experimental value for landfill fire samples. • Octanol-air model was better for predicting PCBs partition constant and only in the case of landfill fire was appropriate for PAHs partition. • Results are suggesting that PAHs partition is close to an adsorption process while PCBs and nal kanes partition is closer to an absorption proces

Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera

Understanding the dynamics of the selection of influenza A immune escape variants by serum antibody is critical for designing effective vaccination programs for animals, especially poultry where large populations have a short generation time and may be vaccinated with high frequency. In this report, immune-escape mutants of A/turkey/New York/4450/1994 H7N2 low pathogenic avian influenza virus, were selected by serially passaging the virus in the presence of continuously increasing concentrations of homologous chicken polyclonal sera. Amino acid mutations were identified by sequencing the parental hemagglutinin (HA) gene and every 10 passages by both Sanger and deep sequencing, and the antigenic distance of the mutants to the parent strain was determined. Progressively, a total of five amino acid mutations were observed over the course of 30 passages. Based on their absence from the parental virus with deep sequencing, the mutations appear to have developed de novo. The antigenic distance between the selected mutants and the parent strain increased as the number of amino acid mutations accumulated and the concentration of antibodies had to be periodically increased to maintain the same reduction in virus titer during selection. This selection system demonstrates how H7 avian influenza viruses behave under selection with homologous sera, and provides a glimpse of their evolutionary dynamics, which can be applied to developing vaccination programs that maximize the effectiveness of a vaccine over time

Mutations in the haemagglutinin protein and their effect in transmission of highly pathogenic avian influenza (HPAI) H5N1 virus in sub-optimally vaccinated chickens

Background Transmission of highly pathogenic avian influenza (HPAI) viruses in poultry flocks is associated with huge economic losses, culling of millions of birds, as well as human infections and deaths. In the cases where vaccination against avian influenza is used as a control measure, it has been found to be ineffective in preventing transmission of field strains. Reports suggest that one of the reasons for this is the use of vaccine doses much lower than the ones recommended by the manufacturer, resulting in very low levels of immunity. In a previous study, we selected for immune escape mutants using homologous polyclonal sera and used them as vaccines in transmission experiments. We concluded that provided a threshold of immunity is reached, antigenic distance between vaccine and challenge strains due to selection need not result in vaccine escape. Here, we evaluate the effect that the mutations in the haemagglutinin protein of our most antigenically-distant mutant may have in the transmission efficiency of this mutant to chickens vaccinated against the parent strain, under sub-optimal vaccination conditions resembling those often found in the field. Methods In this study we employed reverse genetics techniques and transmission experiments to examine if the HA mutations of our most antigenically-distant mutant affect its efficiency to transmit to vaccinated chickens. In addition, we simulated sub-optimal vaccination conditions in the field, by using a very low vaccine dose. Results We find that the mutations in the HA protein of our most antigenically-distant mutant are not enough to allow it to evade even low levels of vaccination-induced immunity. Discussion Our results suggest that – for the antigenic distances we investigated – vaccination can reduce transmission of an antigenically-distant strain compared to the unvaccinated groups, even when low vaccine doses are used, resulting in low levels of immunity.</p

Post-Vaccination Neutralization Responses to Omicron Sub-Variants

Background: The emergence of the Omicron variant (B.1.1.529), which correlated with dramatic losses in cross-neutralization capacity of post-vaccination sera, raised concerns about the effectiveness of COVID-19 vaccines against infection and disease. Several clinically relevant sub-variants subsequently emerged rapidly. Methods: We evaluated published and pre-print studies reporting sub-variant specific reductions in cross-neutralization compared to the prototype strain of SARS-CoV-2 and between sub-variants. Median fold-reduction across studies was calculated by sub-variant and vaccine platform. Results: Among 178 studies with post-vaccination data, after primary vaccination the sub-variant specific fold-reduction in neutralization capacity compared to the prototype antigen varied widely, from median 4.2-fold for BA.3 to 40.1-fold for BA.2.75; in boosted participants fold-reduction was similar for most sub-variants (5.3-fold to 7.0-fold); however, a more pronounced fold-change was observed for sub-variants related to BA.4 and BA.5 (10.4-fold to 14.2-fold). Relative to BA.1, the other Omicron sub-variants had similar neutralization capacity post-primary vaccination (range median 0.8-fold to 1.1-fold) and post-booster (0.9-fold to 1.4-fold) except for BA.4/5-related sub-variants which was higher (2.1-fold to 2.7-fold). Omicron sub-variant-specific responder rates were low post-primary vaccination (range median 28.0% to 65.9%) compared to the prototype (median 100%) but improved post-booster (range median 73.3% to 100%). Conclusions: Fold-reductions in neutralization titers were comparable post-booster except for sub-variants related to BA.4 and BA.5, which had higher fold-reduction. Assessment after primary vaccination was not possible because of overall poor neutralization responses causing extreme heterogeneity. Considering large fold-decreases in neutralization titers relative to the parental strain for all Omicron sub-variants, vaccine effectiveness is very likely to be reduced against all Omicron sub-variants, and probably more so against variants related to BA.4 or BA.5

Immune Escape Mutants of Highly Pathogenic Avian Influenza H5N1 Selected Using Polyclonal Sera: Identification of Key Amino Acids in the HA Protein

<div><p>Evolution of Avian Influenza (AI) viruses – especially of the Highly Pathogenic Avian Influenza (HPAI) H5N1 subtype – is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks) is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an <i>in vitro</i> selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1), using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA) protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.</p></div

Summary of amino acid mutations found in the HA protein of CVI P42, and their locations in respect to antigenically-relevant regions.

<p>Amino acid numbers are shown according to A/turkey/Turkey/1/05 (H5N1) and the reference strain A/goose/Guangdong/1/96 (H5N1) numbering (the latter numbering is used throughout the report unless stated otherwise). Mutations found in common between CVI and FLI isolates are shown in bold. The locations of amino acid substitutions in respect to antigenically relevant regions – according to Duvvuri, <i>et al</i>., (2009) – are shown in the last column (where available). AS: Antigenic Site; PS Site: Positive Selection Site; AP: Antigenic Peptide; RBS: Receptor Binding Site; NA: Not Assigned a function.</p