ABSTRACT Background Patients receiving anti-TNF treatment for chronic inflammatory disease display impaired antibody responses, but it remains unclear how immune memory formation is affected. We evaluated antibody responses and memory B cells (Bmem) after COVID-19 booster vaccination in inflammatory bowel disease (IBD) patients receiving anti-TNF treatment. Methodology Blood was sampled at baseline, 1, and 6 months after WH1/BA.5 bivalent or XBB.1.5 monovalent vaccination from 27 IBD patients receiving intravenous anti-TNF and 44 controls. Neutralizing antibodies were measured using an infectious virus assay. SARS-CoV-2 spike receptor binding domain (RBD)-specific serum IgG was quantified by ELISA, and RBD-specific Bmem were immunophenotyped by flow cytometry using recombinant proteins from ancestral, Omicron BA.1, BA.5, XBB.1.5, and JN.1 variants. Results Serum IgG to vaccine RBD and neutralizing antibodies in patients increased pre to 1 month post-vaccination, but were lower than controls. Ancestral-, BA.5- and XBB.1.5-specific Bmem increased after vaccination but were significantly lower in patients than controls. Within RBD-specific Bmem, frequencies of recently activated CD21 lo cells were increased after vaccination, and were higher in patients than controls. Fewer antigen-specific Bmem in patients expressed IgG4, and more expressed IgG3 or IgD following vaccination. Following vaccination, more RBD-specific Bmem recognized multiple viral variants. However, patients had fewer Bmem that could bind to subvariants than controls. Conclusion Antibody and Bmem responses to COVID-19 booster vaccination in anti-TNF-treated IBD patients displayed reduced capacity, durability and cross-reactivity, suggesting impaired immune memory for protection against breakthrough infection. This supports the recommendation for annual booster vaccination to prevent severe disease and viral spread. Highlights IBD patients on anti-TNF biologics mount antibody responses and form vaccine-specific memory B cells following WH1/BA.5 bivalent or XBB.1.5 monovalent mRNA COVID-19 booster vaccination. Vaccine-specific antibody responses and memory B cell formation in patients are significantly lower than in controls at 1- and 6 months after vaccination, indicative of poorer peak response and durability. Despite multiple vaccinations, vaccine-specific memory B cells from patients display phenotypic alterations and reduced recognition of multiple SARS-CoV-2 Omicron subvariants, potentially a consequence of impaired germinal center responses in patients.

Abstract Objectives Immunocompromised hosts have reduced immune responses to COVID‐19 vaccination, and more severe disease. Antibody responses correlate with protection but markers of immunity vary across a spectrum of immunocompromise. We compared serologic and cellular responses following Ancestral COVID‐19 vaccines in healthy controls (HC), people with HIV (PWH) and lung transplant (LTx) recipients. Methods Anti‐spike receptor binding domain (RBD) IgG, neutralising antibodies (nAb) and T‐cell responses were assessed one‐month post‐dose 2 and dose 3 of Ancestral COVID‐19 vaccination in HC, PWH and LTx. NAb responses to Ancestral, Delta and Omicron BA.2 and BA.5 variants were assessed. Results Twenty‐nine HC, 21 PWH and 12 LTx recipients were included. PWH demonstrated lower anti‐RBD‐IgG responses (median post‐dose 3: 80.3 μg mL−1 vs 43.3 μg mL−1, P = 0.03) to mRNA COVID‐19 vaccination than HC, while LTx recipients displayed diminished responses following any vaccine (15.3 μg mL−1 vs 74.0 μg mL−1, P = 0.01). Dose 3 increased anti‐RBD‐IgG concentrations and nAb responses in HC and PWH, though Omicron variant neutralisation was attenuated. LTx recipients mounted limited nAb responses. PWH and HC had no difference in nAb responses for Ancestral (median 1738 vs 486.2, P > 0.99) or BA.5 variants (median 34.0 vs 67.9, P > 0.99). Compared with HC, PWH and LTx demonstrated reduced frequencies of SARS‐CoV‐2‐specific memory T cells and a reduced functional memory T‐cell response in LTx. Conclusion Although Dose 3 was beneficial, LTx recipients demonstrated lower serological responses than HC, while reductions were modest in PWH. Immunocompromised groups had reduced but detectable SARS‐CoV‐2‐specific T‐cell responses, demonstrating the utility of COVID‐19 vaccination despite poorer serological responses.

IntroductionSevere COVID-19 disease is characterised by a state of hyperinflammation. As key producers of inflammatory mediators in blood, altered inflammatory activity of monocytes within some individuals may contribute to adverse disease outcomes.MethodsAnalysis of monocyte activity under settings of infection and inflammation can be challenged by activation-induced shedding of monocyte receptors traditionally used to identify monocyte subsets. Here, we utilised alternative, more robust immunophenotyping approaches to investigate the impact of COVID-19 infection on monocyte phenotype and inflammatory status.ResultsImmunophenotype analysis of cryopreserved peripheral blood mononuclear cells from unvaccinated, previously COVID-19 naive individuals (median age 43 years, range 21-95, n=42) with a PCR-confirmed acute COVID-19 infection indicated expansion of a novel population of CD14lowCD16- monocytes as compared to control individuals (adjusted p value <0.001) which was more pronounced in individuals with severe disease presentation (p=0.004 for mild vs severe disease). Expansion of this population during acute COVID-19 was confirmed using an alternative, CD14 and CD16-independent strategy for identifying monocyte subsets and was termed infection associated monocytes (IAM). IAM were refractory to ex vivo stimulation with lipopolysaccharide (LPS) and their proportion correlated with plasma levels of TNF and CXCL10 (p<0.05 for both). Monocyte subsets from individuals with acute COVID-19 exhibited reduced basal and LPS-stimulated production of inflammatory cytokines including IL-1β, TNF and IL-6, indicative of an inert monocyte state. Expansion of IAM and impaired inflammatory activity persisted for up to 3 months after acute COVID-19 infection.DiscussionCOVID-19 is associated with expansion of a novel subset of monocytes with impaired inflammatory activity that persist for at least 3 months after acute infection. Whether this population is uniquely expanded by COVID-19, and the long term implications of its persistence post-acute disease, remain to be defined.

Background: Immunocompromised people, including those with Inborn Errors of Immunity (IEI), are at increased risk of severe disease from viral infections. Therefore, regular booster vaccinations are recommended for SARS-CoV-2 and influenza, but it is unclear if these elicit protective immunity. Objective: Comprehensive evaluation of adaptive immune responses, including SARS-COV-2 specific antibodies, memory B- (Bmem) and memory T-cells (Tmem), to COVID-19 vaccination in IEI patients. Methods: Blood samples were collected at 1-month post doses 2 and 3 of the ancestral COVID-19 vaccine, SARS-CoV-2 neutralizing antibodies (NAb) and Spike receptor binding domain (RBD) specific IgG were determined in 25 IEI patients and 29 controls. Ancestral Spike specific Tmem, and ancestral and Omicron subvariant RBD-specific Bmem were evaluated with flow cytometry. Results: After dose 2, IEI patients had significantly lower Nab, RBD-specific IgG and Bmem against ancestral and Omicron subvariants. Third dose vaccination boosted NAb, IgG and Bmem levels, but these remained lower than healthy controls. Especially IgG1+ Bmem were lower in the IEI patients, while they carried higher frequencies of CD71+ ancestral RBD-specific Bmem. IEI patients and controls had similar numbers of Spike-specific CD4+ and CD8+ Tmem after both doses. However, patients Tmem had lower CD69 expression and reduced cytokine co-expression. While 9/25 IEI patients did not have NAb after dose 3, all had detectable SARS-CoV-2 specific IgG, Bmem- and/or Tmem. Conclusion: Patients with IEI form lower levels of antibodies and immune memory cells to COVID-19 vaccination than controls. Still, all patients displayed formation of adaptive immune memory. This suggests a beneficial effect of vaccination, and supports the strategy for offering regular booster vaccinations to limit severe COVID-19 in this at-risk population. ### Competing Interest Statement Conflicting Interests: MCvZ, REOH and PMH are inventers on a patent application related to this work. SJB is an employee of and owns stock in BD. All the other authors declare that they have no conflict of interest. ### Funding Statement The work was supported by the Australian Medical Research Future Fund (MRFF, project no. 2016108), The Jeffrey Modell Foundation, and an Allergy and Immunology Foundation of Australasia (AIFA) Primary Immunodeficiency Clinical Research Grant (supported by CSL Behring, Australia). RG thanks the Burnet Institute for supporting a sabbatical at Monash University. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was conducted according to the Declaration of Helsinki and approved by local ethics committees (Alfred Health ethics no. 32/21, Monash University project no. 72794). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript.

Broadly effective vaccines are needed to protect against future pandemics caused by severe acute respiratory syndrome coronavirus (SARS CoV)-like coronaviruses (sarbecoviruses). The development of simple trimeric subunit vaccines based on the Sarbecovirus spike (S) has proven problematic due to the unstable nature of the S trimer. Here we developed clamp-free, highly stable soluble S trimers by truncating the stem helix to maximize yield and covalently linking the 3 monomers via engineered disulfides to increase thermostability. In K18hACE2 mice, covalently linked SARS CoV-2 S trimers elicited >10-fold higher neutralizing antibody (NAb) titres than parental unlinked trimers and protected the mice against viral challenge. A trivalent vaccine formulation comprised of covalently stabilized spikes derived from 3 divergent ACE2-using Sarbecovirus clades elicited broad and potent neutralizing activity in mice. The covalently linked S trimers were stable at 37°C for 112 days and remained intact following lyophilization and storage at ambient temperature for 6 months. This study establishes a framework for producing simple and stable highly immunogenic pan-Sarbecovirus S subunit vaccines that can be stored and distributed in the absence of a cold chain. ### Competing Interest Statement PP and HD are inventors on a patent application related to this work mRNA Victoris

The Australasian Virology Society (AVS) holds premier biennial virology meetings that foster multidisciplinary research and collaboration and promote equity and inclusion of early-career researchers. The 12th AVS meeting (AVS12), convened by M. Tate, J. Fraser, and G. Moseley, was held from 2 to 5 December 2024 on Dja Dja Wurrung country at the RACV Goldfields Resort in Creswick, Victoria, Australia. In this report, we give a brief overview of the history of AVS and outline the current and developing priorities for the society. We provide a summary of the insightful panel discussions held to address career development and Indigenous virology, highlight the presentations given by international plenary speakers Joe Grove and Chantal Abergel, and celebrate the recipients of the numerous awards.

Currently, the elimination of HCV relies on diagnostics to identify active infection, drugs to treat infected people, and harm reduction approaches to minimize the risk of infection. However, the incidence of new HCV infections continues to outpace the number of curative treatments in many countries; only 11 countries are on track to achieve the World Health Organization's 2030 elimination targets. The missing public health tool to reduce incidences of hepatitis C is a vaccine that prevents HCV infection. The ideal HCV vaccine should induce both cellular and humoral immunity, and such immunity must be sufficiently broad to be effective against at least a majority of circulating genotypes. Neutralizing antibodies targeting multiple distinct epitopes within the E1E2 glycoprotein region and CD4+ and CD8+ T cells targeting multiple regions of the virus should be generated by future vaccines to minimize the risk of immune escape and progression to chronic infection. A successful vaccine will substantially increase the number of countries able to achieve elimination and reduce the cost of elimination.1 Multiple obstacles have slowed the development of a hepatitis C vaccine, including its high genomic diversity, which exceeds that of HIV, difficulty in directly culturing the virus isolated from infected people in cell lines limiting the available strains for characterization, the uncertainty of the exact correlates of immune protection including the relative importance of cellular responses versus neutralizing antibodies in protection, and the lack of immune-competent small animal challenge models to test the efficacy of vaccine candidates. To overcome these obstacles, novel technologies and methodologies have been developed and have led to the discovery of multiple promising candidate vaccines in preclinical studies over the past 5 years, with many showing an ability to elicit broadly neutralizing antibodies in small animals as assessed in in vitro assays. The chimpanzee is the only immune-competent animal model that can recapitulate HCV infection in humans and has been invaluable for our understanding of the virus. However, the chimpanzee is no longer available for biomedical research. The discovery that the fulminant hepatitis C isolate JFH1 was able to replicate in Huh 7.5 cells provided the first system in which to study the complete viral life cycle in vitro as well as evaluate neutralizing antibodies to HCV in infected people and vaccine-derived immune serum using virions that more closely resemble those produced in natural infection. To overcome the inability to readily grow intact isolates from other genotypes and subtypes of HCV, an alternative approach was adopted. Intergenotypic recombinant viruses were generated in which the Core-NS2 region of representative isolates of each major genotype of HCV was chimerized onto the backbone of the JFH1 nonstructural region. In most cases, adaptive mutations were required to confer chimeric viruses with the ability to replicate in Huh 7.5 liver cell lines.2 These cell culture–derived chimeric viruses allowed the assessment of neutralizing antibodies present in immune serum to cross-neutralize different genotypes and subtypes of HCV.2 However, the introduction of these viruses into primary liver cells requires further adaptive mutations for efficient replication, and so there is a lack of viral genetic unity between in vitro and in vivo systems. More recently, Bankwitz et al3 have developed a panel of 13 chimeric HCV viruses representing 5 genotypes and 9 distinct subtypes without adaptive mutations and could be grouped into 6 distinct neutralization clusters. While useful for short-term experiments, this virus panel is not genetically stable in the long term, acquiring adaptive mutations for sustained growth, even in cell culture. The immune-deficient chimeric urokinase-type plasminogen activator (uPA)-severe combined immunodeficiency (SCID) mouse (uPA-SCID) can be reconstituted with primary human liver cells and supports the replication of HCV in vivo4. Passive transfer of immunoglobulins into these reconstituted mice before challenge can determine the capacity of immune serum to achieve protection.5,6 In these studies, the viral inoculum used was immune serum from a chronically infected patient, patient H. Using a J6/JFH1 chimeric virus, Lindenbach et al showed that cell culture–derived virus could also infect human liver reconstituted uPA-SCID mice.7 However, an extension of the utility of the human liver-uPA-SCID model beyond supporting infection with cell culture–derived genotype 2a viruses JFH1 and J6/JFH1 has not to date been achieved but is critical to validate the breadth of in vitro neutralization potency of vaccine-induced antisera in an in vivo model that recapitulates bona vide HCV replication in human liver cells. An article by Collignon et al8 has established a system where the neutralization activity of monoclonal antibodies or immune serum to hepatitis C can be evaluated using the same viruses in vitro and in vivo. By growing JFH1-based Core-NS2 genotype 1–6 cell culture-adapted recombinant viruses in human liver uPA-SCID mice, they identified mouse adaptive mutations within the E1E2 region and reverse engineered these into the parental genome. These adaptive mutations were genetically stable in the case of genotype 1–5 variants, while some strains acquired additional adaptive mutations when cultured in Huh 7.5 cells. Importantly, the viruses showed robust replication with an ability to passage these viruses. Reverse-engineered mouse-adapted variant viruses or genomic RNA of genotypes 1a, 1b, 2b, 2c, 3a, 4a, 5a, and 6a were then used to infect human liver-uPA-SCID mice where they achieved high titer infection of 107–108 genome equivalents/mL and sustained infection beyond 4 weeks. Because these viruses contain mutations in the E1E2 region, well-defined broadly neutralizing monoclonal antibodies were used to assess changes in their sensitivity to neutralization compared to the parental viruses, with some increases and decreases in sensitivity to neutralization noted. To confirm that mouse-adapted virus variants can be used to assess neutralization in vivo in the human liver-uPA-SCID mouse, proof-of-concept experiments using well-characterized potent broadly neutralizing monoclonal antibody AR4A were performed. Mice received an infusion of AR4A and were then challenged with selected mouse-adapted virus variants. No virus was detected in the passively immunized mice, while control animals were able to maintain virus replication for at least 8 weeks, demonstrating that AR4A neutralizes these recombinant viruses with defined adaptive mutations both in vitro and in vivo. The development of this system by Collignon et al could be a valuable tool for HCV vaccine development by further de-risking and prioritizing promising vaccines in preclinical development. Researchers will be able to evaluate immune sera in cell culture–based neutralization assays and then verify the neutralizing activity of immune serum, using the same virus panel, in human-liver-uPA-SCID challenge experiments that more closely resemble the virus replication cycle and the viral-lipoprotein content observed in natural infection. The ability to test immune serum in this system could be essential for down selection and prioritization of the most promising vaccine candidates and will bridge a gap between preclinical and clinical testing. The remaining limitations of using this panel of JFH1-based Core-NS2 genotype 1–6 adapted recombinant viruses in human liver uPA-SCID mouse model are the lack of a functional immune system and the inability to evaluate whether cellular immune responses generated by vaccine candidates contribute to protection. While this panel of chimeric viruses includes isolates of all major genotypes of HCV, only 3 of the 6 neutralization clusters identified by Bankwitz et al3 are presented, and so there remains a gap in the full assessment of the breadth of neutralization. Finally, the range in neutralization sensitivity of this panel of viruses needs to be considered in the evaluation of the neutralizing activity of immune serum. Here, a panel of antigenically and genetically diverse hepatitis C glycoproteins that can be pseudotyped into retroviral particles (HCVpp) may prove valuable. This panel of 15 glycoproteins, mainly derived from genotype 1a glycoproteins,9 exhibit various sensitivities to neutralization from tier 1 (easiest to neutralize) to tier 4 (most difficult to neutralize). While HCVpp is a reliable model to test for entry-blocking antibodies, there are subtle differences in the entry process between HCVpp and HCV virions derived from human hepatocytes due to many factors, including different arrangements and content of glycoproteins E1E2 on their surface, different lipoprotein content, and receptor dependencies. Recently, there has been significant optimism in the field of HCV vaccine development as many promising candidates have been described as having the ability to generate humoral and/or cellular immune responses. Furthermore, mRNA-based vaccines offer a new platform to induce a protective immune response against HCV that may be simpler and cheaper to develop. In addition, the development of a human challenge model for hepatitis C is progressing that will accelerate the clinical development of HCV vaccines by allowing the assessment of the most promising candidates in humans with intact immune systems to assess the level and type of protection afforded.10 Preclinical screening of promising candidates in the system of Collignon and colleagues could be an effective means by which to select the most promising candidates for human challenge studies. This is an exciting time for HCV vaccine development. Combined with highly effective drugs, the successful addition of an effective vaccine would put the elimination of HCV-induced viral hepatitis within reach.

Booster vaccinations are recommended to improve protection against severe disease from SARS-CoV-2 infection. With primary vaccinations involving various adenoviral vector and mRNA-based formulations, it remains unclear if these differentially affect the immune response to booster doses. We examined the effects of homologous (mRNA/mRNA) and heterologous (adenoviral vector/mRNA) vaccination on antibody and memory B cell (Bmem) responses against ancestral and Omicron subvariants. Healthy adults who received primary BNT162b2 (mRNA) or ChAdOx1 (vector) vaccination were sampled 1-month and 6-months after their 2nd and 3rd dose (homologous or heterologous) vaccination. Recombinant spike receptor-binding domain (RBD) proteins from ancestral, Omicron BA.2 and BA.5 variants were produced for ELISA-based serology, and tetramerized for immunophenotyping of RBD-specific Bmem. Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1+. IgG4+ Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5-20% after dose 3, whereas heterologous boosting did not elicit IgG4+ Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster. A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4+ Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.

Objectives. Despite vaccination strategies, people with chronic kidney disease, particularly kidney transplant recipients (KTRs), remained at high risk of poor COVID-19 outcomes. We assessed serological responses to the three-dose COVID-19 vaccine schedule in KTRs and people on dialysis, as well as seroresponse predictors and the relationship between responses and breakthrough infection. Methods. Plasma from 30 KTRs and 17 people receiving dialysis was tested for anti-Spike receptor binding domain (RBD) IgG and neutralising antibodies (NAb) to the ancestral and Omicron BA.2 variant after Doses 2 and 3 of vaccination. Results. After three doses, KTRs achieved lower anti-Spike RBD IgG levels (P < 0.001) and NAb titres than people receiving dialysis (P = 0.002). Seropositive cross-reactive Omicron neutralisation levels were achieved in 11/27 (40.7%) KTRs and 11/14 (78.6%) dialysis recipients. ChAdOx1/viral-vector vaccine type, higher mycophenolate dose (> 1 g per day) and lower absolute B-cell counts predicted poor serological responses in KTRs. ChAdOx-1 vaccine type and higher monocyte counts were negative predictors in dialysis recipients. Among ancestral NAb seroresponders, higher NAb levels positively correlated with higher Omicron neutralisation (R = 0.9, P < 0.001). More KTRs contracted SARS-CoV-2 infection (14/30; 47%) than dialysis recipients (5/17; 29%) and had more severe disease. Those with breakthrough infections had significantly lower median interdose incremental change in anti-Spike RBD IgG and ancestral NAb titres. Conclusion. Serological responses to COVID-19 vaccines in KTRs lag behind their dialysis counterparts. KTRs remained at high risk of breakthrough infection after their primary vaccination schedule underlining their need for booster doses, strict infection prevention measures and close surveillance.

Background and aimsIn individuals highly exposed to hepatitis C virus (HCV), reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection.MethodsBroad neutralising antibodies (BnAbs) and Envelope 2 (E2)-specific memory B cell (MBCs) responses were examined longitudinally in 15 subjects with varied reinfection outcomes.ResultsBnAb responses were associated with MBC recall, but not with reinfection clearance. Strong evidence of antigen imprinting was found, and the B cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection.ConclusionsMBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development.

BackgroundSARS-CoV-2 transmission and COVID-19 disease severity is influenced by immunity from natural infection and/or vaccination. Population-level immunity is complicated by the emergence of viral variants. Antibody Fc-dependent effector functions are as important mediators in immunity. However, their induction in populations with diverse infection and/or vaccination histories and against variants remains poorly defined.MethodsWe evaluated Fc-dependent functional antibodies following vaccination with two widely used vaccines, AstraZeneca (AZ) and Sinovac (SV), including antibody binding of Fc gamma-receptors and complement-fixation in vaccinated Brazilian adults (n = 222), some of who were previously infected with SARS-CoV-2, as well as adults with natural infection only (n = 200). IgG, IgM, IgA, and IgG subclasses were also quantified.ResultsAZ induces greater Fc gamma-receptor-binding (types I, IIa, and IIIa/b) antibodies than SV or natural infection. Previously infected individuals have significantly greater vaccine-induced responses compared to na & iuml;ve counterparts. Fc gamma-receptor-binding is highest among AZ vaccinated individuals with a prior infection, for all receptor types, and substantial complement-fixing activity is only seen among this group. SV induces higher IgM than AZ, but this does not drive better complement-fixing activity. Some SV responses are associated with subject age, whereas AZ responses are not. Importantly, functional antibody responses are well retained against the Omicron BA.1 S protein, being best retained for Fc gamma-receptor-1 binding, and are higher for AZ than SV.ConclusionsHybrid immunity, from combined natural exposure and vaccination, generates strong Fc-mediated antibody functions which may contribute to immunity against evolving SARS-CoV-2 variants. Understanding determinants of Fc-mediated functions may enable future vaccines with greater efficacy against different variants.

BackgroundHepatitis C virus (HCV) infections are more prevalent in people who inject drugs (PWID) who often experience additional health risks. HCV induces inflammation and immune alterations that contribute to hepatic and non-hepatic morbidities. It remains unclear whether curative direct acting antiviral (DAA) therapy completely reverses immune alterations in PWID.MethodsPlasma biomarkers of immune activation associated with chronic disease risk were measured in HCV-seronegative (n=24) and HCV RNA+ (n=32) PWID at baseline and longitudinally after DAA therapy. Adjusted generalised estimating equations were used to assess longitudinal changes in biomarker levels. Comparisons between community controls (n=29) and HCV-seronegative PWID were made using adjusted multiple regression modelling.ResultsHCV-seronegative PWID exhibited significantly increased levels of inflammatory biomarkers including soluble (s) TNF-RII, IL-6, sCD14 and sCD163 and the diabetes index HbA1c as compared to community controls. CXCL10, sTNF-RII, vascular cell adhesion molecule-1 and lipopolysaccharide binding protein (LBP) were additionally elevated in PWID with viremic HCV infection as compared to HCV- PWID. Whilst curative DAA therapy reversed some biomarkers, others including LBP and sTNF-RII remained elevated 48 weeks after HCV cure.ConclusionElevated levels of inflammatory and chronic disease biomarkers in PWID suggest an increased risk of chronic morbidities such as diabetes and cardiovascular disease. HCV infection in PWID poses an additional disease burden, amplified by the incomplete reversal of immune dysfunction following DAA therapy. These findings highlight the need for heightened clinical surveillance of PWID for chronic inflammatory diseases, particularly those with a history of HCV infection.

Background Prevalence of hepatitis C virus (HCV) antibody (Ab) on dried blood spot (DBS) samples in the Australian Needle and Syringe Program Survey (ANSPS) decreased nationally from 57% in 2015 to 32% in 2022. We aimed to investigate potential explanations for this decline. Methods Changes in DBS HCV Ab prevalence were investigated by redefining positive cases as those with those with either a positive HCV Ab test result or a self-reported history of ever having HCV treatment (modified prevalence), examining HCV Ab prevalence by birth and age cohorts, and assessing trends in key risk behaviours. Results Overall prevalence of DBS HCV Ab declined rapidly and significantly from 57% in 2015 to 32% in 2022 (p<0.001) however modified HCV Ab prevalence remained stable over time (85% and 88% in 2015 and 2022, respectively, p=0.357). The proportion of participants with negative HCV Ab and self-reported HCV infection increased from 20% in 1995 to 40% in 2022 (p<0.001) and the proportion with negative HCV Ab and lifetime HCV treatment increased from 3% in 1999 to 67% in 2022 (p<0.001). We also observed a decreasing trend in DBS HCV Ab prevalence in all birth and age cohorts with a noticeable acceleration in the decline commensurate with the advent of HCV DAA treatment. A long-term decreasing trend was also observed for key risk behaviours (p<0.001) however the short-term trend was not significant for recent receptive syringe sharing. Conclusion The temporal decline in HCV Ab prevalence appears related to reduced sensitivity of DBS HCV Ab detection with viral clearance following treatment. Since 2016, HCV treatment uptake has increased markedly including among people who inject drugs. In this context, continuing to monitor HCV Ab prevalence by DBS testing is problematic, with a shift to surveillance of active infection the most relevant to guide policy and practice in this setting.

BACKGROUND:The RTS,S malaria vaccine is currently recommended for children aged 5-6 months in regions with moderate-to-high Plasmodium falciparum transmission. However, vaccination only confers 55% efficacy over 12 months and wanes within 18 months. The immunological mechanisms of RTS,S-mediated immunity are poorly understood; therefore, we aimed to identify antibody response types associated with protection against malaria in children vaccinated with RTS,S. METHODS:In this post-hoc analysis, we evaluated antibody responses in 737 children aged 1-4 years vaccinated with RTS,S in a phase 2b clinical trial conducted in Mozambique in 2003. We evaluated all available samples collected from children 30 days after the three-dose vaccination schedule at study month 3 (M3; n=737 available of 803 children allocated to receive RTS,S). For comparison, we tested a subset of samples collected before vaccination at study month 0 (M0; n=50) and from children in the control vaccine group (M0 n=25; M3 n=99). We quantified the induction of antibodies to different regions of the vaccine antigen that function by fixing serum complement proteins and binding to Fcγ receptors (FcγRs; FcγRI, FcγRIIa, and FcγRIII) expressed on immune cells as potential mechanisms of immunity. FINDINGS:Functional antibody responses to the C-terminal region of the vaccine antigen, circumsporozoite protein (CSP), were associated with a reduced risk of malaria (C1q p=0·0060, FcγRIIa p=0·014, and FcγRIII p=0·019). These associations remained significant in male participants when the analyses were stratified by sex (C1q p=0·012, FcγRI p=0·023, FcγRIIa p=0·0070, and FcγRIII p=0·0080). IgA to the central repeat (p=0·0010) and C-terminal (p=0·0040) regions of CSP were also associated with protection. We show that IgA can bind FcαRI and mediate opsonic phagocytosis using a serum pool and monoclonal antibodies. Multiparameter analysis using machine-learning methods suggest that IgA, complement fixation, and FcγRI binding were most predictive of protection against malaria (hazard ratio <1) and suggested that associations differed between male and female participants. INTERPRETATION:We provide evidence that functional antibody responses mediated by IgG and IgA are associated with protection against malaria in young children vaccinated with RTS,S, and suggest potential differences in the correlates of immunity between males and females. These findings reveal new avenues that could be used to achieve malaria vaccines with higher efficacy. FUNDING:National Health and Medical Research Council, Australia, and Thrasher Research Fund.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.

Following the COVID-19 pandemic, novel vaccines have successfully reduced severe disease and death. Despite eliciting lower antibody responses, adenoviral vector vaccines are nearly as effective as mRNA vaccines. Therefore, protection against severe disease may be mediated by immune memory cells. We here evaluated plasma antibody and memory B cells (Bmem) targeting the SARS-CoV-2 Spike receptor-binding domain (RBD) elicited by the adenoviral vector vaccine ChAdOx1 (AstraZeneca), their capacity to bind Omicron subvariants, and compared this to the response to mRNA BNT162b2 (Pfizer-BioNTech) vaccination. Whole blood was sampled from 31 healthy adults pre-vaccination and 4 weeks after dose one and dose two of ChAdOx1. Neutralizing antibodies (NAb) against SARS-CoV-2 were quantified at each time point. Recombinant RBDs of the Wuhan-Hu-1 (WH1), Delta, BA.2, and BA.5 variants were produced for ELISA-based quantification of plasma IgG and incorporated separately into fluorescent tetramers for flow cytometric identification of RBD-specific Bmem. NAb and RBD-specific IgG levels were over eight times lower following ChAdOx1 vaccination than BNT162b2. In ChAdOx1-vaccinated individuals, median plasma IgG recognition of BA.2 and BA.5 as a proportion of WH1-specific IgG was 26% and 17%, respectively. All donors generated resting RBD-specific Bmem, which were boosted after the second dose of ChAdOx1 and were similar in number to those produced by BNT162b2. The second dose of ChAdOx1 boosted Bmem that recognized VoC, and 37% and 39% of WH1-specific Bmem recognized BA.2 and BA.5, respectively. These data uncover mechanisms by which ChAdOx1 elicits immune memory to confer effective protection against severe COVID-19.

ABSTRACT Background Booster vaccinations are recommended to improve protection against severe disease from SARS-CoV-2 infection. With primary vaccinations involving various adenoviral vector and mRNA-based formulations, it remains unclear if these differentially affect the immune response to booster doses. We here examined the effects of homologous (mRNA/mRNA) and heterologous (adenoviral vector/mRNA) vaccination on antibody and memory B cell (Bmem) responses against ancestral and Omicron subvariants. Methods Healthy adults who received primary BNT162b2 (mRNA) (n=18) or ChAdOx1 (vector) (n=25) vaccination were sampled 1-month and 6-months after their 2nd and 3rd dose (homologous or heterologous) vaccination. Recombinant spike receptor-binding domain (RBD) proteins from ancestral, Omicron BA.2 and BA.5 variants were produced for ELISA-based serology, and tetramerized for immunophenotyping of RBD-specific Bmem. Results Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1 + . IgG4 + Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5-20% after dose 3, whereas heterologous boosting did not elicit IgG4 + Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster. Conclusion A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4 + Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.