Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

COVID-19 seroprevalence cohort survey among health care workers and their household members in Kinshasa, DR Congo, 2020-2022.

INTRODUCTION: Serological surveys offer the most direct measurement to define the immunity status for numerous infectious diseases, such as COVID-19, and can provide valuable insights into understanding transmission patterns. This study describes seroprevalence changes over time in the context of the Democratic Republic of Congo, where COVID-19 case presentation was apparently largely oligo- or asymptomatic, and vaccination coverage remained extremely low. METHODS: A cohort of 635 health care workers (HCW) from 5 health zones of Kinshasa and 670 of their household members was interviewed and sampled in 6 rounds between July 2020 and January 2022. At each round, information on risk exposure and a blood sample were collected. Serology was defined as positive when binding antibodies against SARS-CoV-2 spike and nucleocapsid proteins were simultaneously present. RESULTS: The SARS-CoV-2 antibody seroprevalence was high at baseline, 17.3% (95% CI 14.4-20.6) and 7.8% (95% CI 5.5-10.8) for HCW and household members, respectively, and fluctuated over time, between 9% and 62.1%. Seropositivity was heterogeneously distributed over the health zones (p < 0.001), ranging from 12.5% (95% CI 6.6-20.8) in N'djili to 33.7% (95% CI 24.6-43.8) in Bandalungwa at baseline for HCW. Seropositivity was associated with increasing rounds adjusted Odds Ratio (aOR) 1.75 (95% CI 1.66-1.85), with increasing age aOR 1.11 (95% CI 1.02-1.20), being a female aOR 1.35 (95% CI 1.10-1.66) and being a HCW aOR 2.38 (95% CI 1.80-3.14). There was no evidence that HCW brought the COVID-19 infection back home, with an aOR of 0.64 (95% CI 0.46-0.91) of seropositivity risk among household members in subsequent surveys. There was seroreversion and seroconversion over time, and HCW had a lower risk of seroreverting than household members (aOR 0.60 (95% CI 0.42-0.86)). CONCLUSION: SARS-CoV-2 IgG antibody levels were high and dynamic over time in this African setting with low clinical case rates. The absence of association with health profession or general risk behaviors and with HCW positivity in subsequent rounds in HH members, shows the importance of the time-dependent, and not work-related, force of infection. Cohort seroprevalence estimates in a 'new disease' epidemic seem insufficient to guide policy makers for defining control strategies.

Lung ILC2s are activated in BALB/c mice born to immunized mothers despite complete protection against respiratory syncytial virus.

Activated lung ILC2s produce large quantities of IL-5 and IL-13 that contribute to eosinophilic inflammation and mucus production following respiratory syncytial virus infection (RSV). The current understanding of ILC2 activation during RSV infection, is that ILC2s are activated by alarmins, including IL-33, released from airway epithelial cells in response to viral-mediated damage. Thus, high levels of RSV neutralizing maternal antibody generated from maternal immunization would be expected to reduce IL-33 production and mitigate ILC2 activation. Here we report that lung ILC2s from mice born to RSV-immunized dams become activated despite undetectable RSV replication. We also report, for the first time, expression of activating and inhibitory Fcgamma receptors on ILC2s that are differentially expressed in offspring born to immunized versus unimmunized dams. Alternatively, ex vivo IL-33-mediated activation of ILC2s was mitigated following the addition of antibody: antigen immune complexes. Further studies are needed to confirm the role of Fcgamma receptor ligation by immune complexes as an alternative mechanism of ILC2 regulation in RSV-associated eosinophilic lung inflammation.

Seasonal human coronavirus humoral responses in AZD1222 (ChaAdOx1 nCoV-19) COVID-19 vaccinated adults reveal limited cross-immunity.

Background: Immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now widespread; however, the degree of cross-immunity between SARS-CoV-2 and endemic, seasonal human coronaviruses (HCoVs) remains unclear. Methods: SARS-CoV-2 and HCoV cross-immunity was evaluated in adult participants enrolled in a US sub-study in the phase III, randomized controlled trial (NCT04516746) of AZD1222 (ChAdOx1 nCoV-19) primary-series vaccination for one-year. Anti-HCoV spike-binding antibodies against HCoV-229E, HCoV-HKU1, HCoV-OC43, and HCoV-NL63 were evaluated in participants following study dosing and, in the AZD1222 group, after a non-study third-dose booster. Timing of SARS-CoV-2 seroconversion (assessed via anti-nucleocapsid antibody levels) and incidence of COVID-19 were evaluated in those who received AZD1222 primary-series by baseline anti-HCoV titers. Results: We evaluated 2,020/21,634 participants in the AZD1222 group and 1,007/10,816 in the placebo group. At the one-year data cutoff (March 11, 2022) mean duration of follow up was 230.9 (SD: 106.36, range: 1-325) and 94.3 (74.12, 1-321) days for participants in the AZD1222 (n = 1,940) and placebo (n = 962) groups, respectively. We observed little elevation in anti-HCoV humoral titers post study-dosing or post-boosting, nor evidence of waning over time. The occurrence and timing of SARS-CoV-2 seroconversion and incidence of COVID-19 were not largely impacted by baseline anti-HCoV titers. Conclusion: We found limited evidence for cross-immunity between SARS-CoV-2 and HCoVs following AZD1222 primary series and booster vaccination. Susceptibility to future emergence of novel coronaviruses will likely persist despite a high prevalence of SARS-CoV-2 immunity in global populations.

Antibody Response Before and After the Booster Dose of Inactivated Corona Vaccine in Antibody Deficient Patients.

Patients with inborn errors of immunity (IEI) are among the high-risk groups regarding COVID-19. Receiving booster doses (third and fourth) in addition to the standard doses is recommended in these patients. This study investigated the antibody response before and after a booster dose of Sinopharm vaccine in IEI patients.  Thirty patients (>12 years) with antibody deficiencies, referred to Imam Khomeini Hospital and Children's Medical Center in Tehran, were enrolled in this prospective cross-sectional study. All patients were fully vaccinated with the BBIBP-CorV vaccine (2 doses of Sinopharm). Initial measurements of anti-receptor-binding domain (anti-RBD) and anti-nucleocapsid (anti-N) IgG antibody responses were conducted by enzyme-linked immunosorbent assay (ELISA). Subsequently, all patients received a booster dose of the vaccine. Four to six weeks after booster injection, the levels of antibodies were re-evaluated.  Twenty patients with common variable immunodeficiency (CVID), 7 cases with agammaglobulinemia and 3 patients with hyper IgM syndrome were studied. Anti-RBD IgG and anti-N IgG antibodies increased in all patients after the booster. Our results indicated the need of receiving booster doses of the COVID-19 vaccine in patients with antibody deficiencies, even for enhancing humoral immune response specially in patients with CVID.

Impact of elevated IgE in protective immunity and immunopathology of dengue.

Dengue is a mosquito-transmitted infection endemic in tropical and subtropical locations of the world where nearly half of the world's population resides. The disease may present as mild febrile illness to severe and can even be fatal if untreated. There are four genetically related but antigenically distinct dengue virus (DENV) serotypes. Immune responses to DENV infection are in general protective but under certain conditions, they can also aggravate the disease. The importance of the cellular immune responses and the antibody responses involving IgG and IgM has been well-studied. In contrast, not much has been described on the potential role of hypersensitivity reactions involving IgE in dengue. Several studies have shown elevated levels of IgE in patients with dengue fever, but its involvement in the immune response against the virus and disease is unknown. Activation of mast cells (MCs) and basophils mediated through dengue-specific IgE could result in the release of mediators affecting dengue virus infection. The present review explores the relationships between the induction of IgE in dengue virus infection, and the potential role of MCs and basophils, exploring both protective and pathogenic aspects, including antibody-dependent enhancement (ADE) of infection in dengue.

Sex-biased immunogenicity of a mucosal subunit vaccine against SARS-CoV-2 in mice.

Introduction: Current vaccines against COVID-19 administered via parenteral route have limited ability to induce mucosal immunity. There is a need for an effective mucosal vaccine to combat SARS-CoV-2 virus replication in the respiratory mucosa. Moreover, sex differences are known to affect systemic antibody responses against vaccines. However, their role in mucosal cellular responses against a vaccine remains unclear and is underappreciated. Methods: We evaluated the mucosal immunogenicity of a booster vaccine regimen that is recombinant protein-based and administered intranasally in mice to explore sex differences in mucosal humoral and cellular responses. Results: Our results showed that vaccinated mice elicited strong systemic antibody (Ab), nasal, and bronchiole alveolar lavage (BAL) IgA responses, and local T cell immune responses in the lung in a sex-biased manner irrespective of mouse genetic background. Monocytes, alveolar macrophages, and CD103+ resident dendritic cells (DCs) in the lungs are correlated with robust mucosal Ab and T cell responses induced by the mucosal vaccine. Discussion: Our findings provide novel insights into optimizing next-generation booster vaccines against SARS-CoV-2 by inducing spike-specific lung T cell responses, as well as optimizing mucosal immunity for other respiratory infections, and a rationale for considering sex differences in future vaccine research and vaccination practice.

A ferritin nanoparticle vaccine based on the hemagglutinin extracellular domain of swine influenza A (H1N1) virus elicits protective immune responses in mice and pigs.

Introduction: Swine influenza viruses (SIVs) pose significant economic losses to the pig industry and are a burden on global public health systems. The increasing complexity of the distribution and evolution of different serotypes of influenza strains in swine herds escalates the potential for the emergence of novel pandemic viruses, so it is essential to develop new vaccines based on swine influenza. Methods: Here, we constructed a self-assembling ferritin nanoparticle vaccine based on the hemagglutinin (HA) extracellular domain of swine influenza A (H1N1) virus using insect baculovirus expression vector system (IBEVS), and after two immunizations, the immunogenicities and protective efficacies of the HA-Ferritin nanoparticle vaccine against the swine influenza virus H1N1 strain in mice and piglets were evaluated. Results: Our results demonstrated that HA-Ferritin nanoparticle vaccine induced more efficient immunity than traditional swine influenza vaccines. Vaccination with the HA-Ferritin nanoparticle vaccine elicited robust hemagglutinin inhibition titers and antigen-specific IgG antibodies and increased cytokine levels in serum. MF59 adjuvant can significantly promote the humoral immunity of HA-Ferritin nanoparticle vaccine. Furthermore, challenge tests showed that HA-Ferritin nanoparticle vaccine conferred full protection against lethal challenge with H1N1 virus and significantly decreased the severity of virus-associated lung lesions after challenge in both BALB/c mice and piglets. Conclusion: Taken together, these results indicate that the hemagglutinin extracellular-based ferritin nanoparticle vaccine may be a promising vaccine candidate against SIVs infection.

High SARS-CoV-2 infection rate in children unvaccinated with COVID-19 vaccine in Changzhou, China, shortly after lifting zero-COVID-19 policy in December 2022.

BACKGROUND: China experienced an overwhelming COVID-19 pandemic from middle December 2022 to middle January 2023 after lifting the zero-COVID-19 policy on December 7, 2022. However, the infection rate was less studied. We aimed to investigate the SARS-CoV-2 infection rate in children shortly after discontinuation of the zero-COVID-19 policy. METHODS: From February 20 to April 10, 2023, we included 393 children aged 8 months to less than 3 years who did not receive COVID-19 vaccination and 114 children aged 3 to 6 years who received inactivated COVID-19 vaccines based on the convenience sampling in this cross-sectional study. IgG and IgM antibodies against nucleocapsid (N) and subunit 1 of spike (S1) of SARS-CoV-2 (anti-N/S1) were measured with commercial kits (Shenzhen YHLO Biotech, China). RESULTS: Of the 393 unvaccinated children (1.5 ± 0.6 years; 52.2% boys), 369 (93.9%) were anti-N/S1 IgG positive. Of the 114 vaccinated children (5.3 ± 0.9 years; 48.2% boys), 112 (98.2%) were anti-N/S1 IgG positive. None of the unvaccinated or vaccinated children was anti-N/S1 IgM positive. The median IgG antibody titers in vaccinated children (344.91 AU/mL) were significantly higher than that in unvaccinated children (42.80 AU/mL) (P < 0.0001). The positive rates and titers of anti-N/S1 IgG had no significant difference between boys and girls respectively. CONCLUSION: Vast majority of children were infected with SARS-CoV-2 shortly after ending zero-COVID-19 policy in China. Whether these unvaccinated infected children should receive COVID-19 vaccine merits further investigation.

Temporal trends in disparities in COVID-19 seropositivity among Canadian blood donors.

BACKGROUND: In Canada's largest COVID-19 serological study, SARS-CoV-2 antibodies in blood donors have been monitored since 2020. No study has analysed changes in the association between anti-N seropositivity (a marker of recent infection) and geographic and sociodemographic characteristics over the pandemic. METHODS: Using Bayesian multi-level models with spatial effects at the census division level, we analysed changes in correlates of SARS-CoV-2 anti-N seropositivity across three periods in which different variants predominated (pre-Delta, Delta and Omicron). We analysed disparities by geographic area, individual traits (age, sex, race) and neighbourhood factors (urbanicity, material deprivation and social deprivation). Data were from 420 319 blood donations across four regions (Ontario, British Columbia [BC], the Prairies and the Atlantic region) from December 2020 to November 2022. RESULTS: Seropositivity was higher for racialized minorities, males and individuals in more materially deprived neighbourhoods in the pre-Delta and Delta waves. These subgroup differences dissipated in the Omicron wave as large swaths of the population became infected. Across all waves, seropositivity was higher in younger individuals and those with lower neighbourhood social deprivation. Rural residents had high seropositivity in the Prairies, but not other regions. Compared to generalized linear models, multi-level models with spatial effects had better fit and lower error when predicting SARS-CoV-2 anti-N seropositivity by geographic region. CONCLUSIONS: Correlates of recent COVID-19 infection have evolved over the pandemic. Many disparities lessened during the Omicron wave, but public health intervention may be warranted to address persistently higher burden among young people and those with less social deprivation.

Assessing the association between antibody status and symptoms of long COVID: A multisite study.

The association between SARS-CoV-2 humoral immunity and post-acute sequelae of COVID-19 (long COVID) remains uncertain. The objective of this population-based cohort study was to assess the association between SARS-CoV-2 seropositivity and symptoms consistent with long COVID. English and Spanish-speaking members ≥ 18 years old with SARS-CoV-2 serologic testing conducted prior to August 2021 were recruited from Kaiser Permanente Southern California and Kaiser Permanente Colorado. Between November 2021 and April 2022, participants completed a survey assessing symptoms, physical health, mental health, and cognitive function consistent with long COVID. Survey results were linked to SARS-CoV-2 antibody (Ab) and viral (RNA) lab results in electronic health records. Weighted descriptive analyses were generated for five mutually exclusive patient groups: (1) +Ab/+RNA; (2) +Ab/- or missing RNA; (3) -Ab/+RNA; (4a) -Ab/-RNA reporting no prior infection; and (4b) -Ab/-RNA reporting prior infection. The proportions reporting symptoms between the +Ab/+RNA and -Ab/+RNA groups were compared, adjusted for covariates. Among 3,946 participants, the mean age was 52.1 years old (SD 15.6), 68.3% were female, 28.4% were Hispanic, and the serologic testing occurred a median of 15 months prior (IQR = 12-18). Three quarters (74.5%) reported having had COVID-19. Among people with laboratory-confirmed COVID-19, there was no association between antibody positivity (+Ab/+RNA vs. -Ab/+RNA) and any symptoms, physical health, mental health, or cognitive function. As expected, physical health, cognitive function, and fatigue were worse, and palpitations and headaches limiting the ability to work were more prevalent among people with laboratory-confirmed prior infection and positive serology (+Ab/+RNA) compared to those without reported or confirmed prior infection and negative serology (-Ab/-RNA/no reported COVID-19). Among people with laboratory-confirmed COVID-19, SARS-CoV-2 serology from practice settings were not associated with long COVID symptoms and health status suggesting limited utility of serology testing for long COVID.

Phase 1, randomized, rater and participant blinded placebo-controlled study of the safety, reactogenicity, tolerability and immunogenicity of H1N1 influenza vaccine delivered by VX-103 (a MIMIX microneedle patch [MAP] system) in healthy adults.

BACKGROUND: The MIMIX platform is a novel microneedle array patch (MAP) characterized by slowly dissolving microneedle tips that deploy into the dermis following patch application. We describe safety, reactogenicity, tolerability and immunogenicity for MIMIX MAP vaccination against influenza. METHODOLOGY: The trial was a Phase 1, exploratory, first-in-human, parallel randomized, rater, participant, study analyst-blinded, placebo-controlled study in Canada. Forty-five healthy participants (18 to 39 years of age, inclusive) were randomized in a 1:1:1 ratio to receive either 15 µg or 7.5 µg of an H1N1 influenza vaccine, or placebo delivered via MIMIX MAP to the volar forearm. A statistician used a computer program to create a randomization scheme with a block size of 3. Post-treatment follow-up was approximately 180 days. Primary safety outcomes included the incidence of study product related serious adverse events and unsolicited events within 180 days, solicited application site and systemic reactogenicity through 7 days after administration and solicited application site erythema and/or pigmentation 14, 28, 56 and 180 days after administration. Immunogenicity outcomes included antibody titers and percentage of seroconversion (SCR) and seroprotection (SPR) rates determined by the hemagglutination inhibition (HAI) assay. Exploratory outcomes included virus microneutralization (MN) titers, durability and breadth of the immune response. The trial was registered with ClinicalTrials.gov, number NCT06125717. FINDINGS: Between July 7, 2022 and March 13, 2023 45 participants were randomized to a treatment group. One participant was lost to follow up in the 15 µg group and 1 participant withdrew from the 7.5 µg dose group. Safety analyses included n = 15 per group, immunogenicity analyses included n = 14 for the 15 µg and 7.5 µg treatment groups and n = 15 for the placebo group. No SAEs were reported in any of the treatment groups. All treatment groups reported solicited local events within 7 days after vaccination, with mild (Grade 1) erythema being the most frequent symptom reported. Other local symptoms reported included mostly mild (Grade 1) induration/swelling, itching, pigmentation, skin flaking, and tenderness. Within 7 days after vaccination, 2 participants (4.4%) reported moderate (Grade 2) erythema, 1 participant (2.2%) reported moderate (Grade 2) induration/swelling, and 1 participant (2.2%) reported moderate (Grade 2) itching. There was an overall reduction in erythema and pigmentation reported on Days 15, 29, 57, and 180 among all treatment groups. Systemic symptoms reported within 7 days after vaccination, included mild (Grade 1) fatigue reported among all treatment groups, and mild (Grade 1) headache reported by 1 participant in the 7.5 µg treatment group. No study drug related severe symptoms were reported in the study. Group mean fold rises in HAI titers ranged between 8.7 and 12-fold, SCRs were >76% and SPRs were >92% for both VX-103 dose groups thereby fulfilling serological criteria established by the EMA and FDA for seasonal influenza vaccines. Longitudinal assessments demonstrate persistence of the immune response through at least Day 180. CONCLUSIONS: The MIMIX MAP platform is safe, well tolerated and elicits robust antibody responses.

Heightened Epstein-Barr virus immunity and potential cross-reactivities in multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) is a likely prerequisite for multiple sclerosis (MS) but the underlying mechanisms are unknown. We investigated antibody and T cell responses to EBV in persons with MS (pwMS), healthy EBV-seropositive controls (HC) and post-infectious mononucleosis (POST-IM) individuals up to 6 months after disease resolution. The ability of EBV-specific T cell responses to target antigens from the central nervous system (CNS) was also investigated. METHODS: Untreated persons with relapsing-remitting MS, POST-IM individuals and HC were, as far as possible, matched for gender, age and HLA-DRB1*15:01. EBV load was determined by qPCR, and IgG responses to key EBV antigens were determined by ELISA, immunofluorescence and Western blot, and tetanus toxoid antibody responses by multiplex bead array. EBV-specific T cell responses were determined ex vivo by intracellular cytokine staining (ICS) and cross-reactivity of in vitro-expanded responses probed against 9 novel Modified Vaccinia Ankara (MVA) viruses expressing candidate CNS autoantigens. RESULTS: EBV load in peripheral blood mononuclear cells (PBMC) was unchanged in pwMS compared to HC. Serologically, while tetanus toxoid responses were unchanged between groups, IgG responses to EBNA1 and virus capsid antigen (VCA) were significantly elevated (EBNA1 p = 0.0079, VCA p = 0.0298) but, importantly, IgG responses to EBNA2 and the EBNA3 family antigens were also more frequently detected in pwMS (EBNA2 p = 0.042 and EBNA3 p = 0.005). In ex vivo assays, T cell responses to autologous EBV-transformed B cells and to EBNA1 were largely unchanged numerically, but significantly increased IL-2 production was observed in response to certain stimuli in pwMS. EBV-specific polyclonal T cell lines from both MS and HC showed high levels of autoantigen recognition by ICS, and several neuronal proteins emerged as common targets including MOG, MBP, PLP and MOBP. DISCUSSION: Elevated serum EBV-specific antibody responses in the MS group were found to extend beyond EBNA1, suggesting a larger dysregulation of EBV-specific antibody responses than previously recognised. Differences in T cell responses to EBV were more difficult to discern, however stimulating EBV-expanded polyclonal T cell lines with 9 candidate CNS autoantigens revealed a high level of autoreactivity and indicate a far-reaching ability of the virus-induced T cell compartment to damage the CNS.

Anti-RBD IgG antibodies from endemic coronaviruses do not protect against the acquisition of SARS-CoV-2 infection among exposed uninfected individuals.

Background: The Coronaviridae family comprises seven viruses known to infect humans, classified into alphacoronaviruses (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1), which are considered endemic. Additionally, it includes SARS-CoV (severe acute respiratory syndrome), MERS-CoV (Middle East respiratory syndrome), and the novel coronavirus SARS-CoV-2, responsible for COVID-19. SARS-CoV-2 induces severe respiratory complications, particularly in the elderly, immunocompromised individuals and those with underlying diseases. An essential question since the onset of the COVID-19 pandemic has been to determine whether prior exposure to seasonal coronaviruses influences immunity or protection against SARS-CoV-2. Methods: In this study, we investigated a cohort of 47 couples (N=94), where one partner tested positive for SARS-CoV-2 infection via real-time PCR while the other remained negative. Plasma samples, collected at least 30 days post-PCR reaction, were assessed using indirect ELISA and competition assays to measure specific antibodies against the receptor-binding domain (RBD) portion of the Spike (S) protein from SARS-CoV-2, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1. Results: IgG antibody levels against the four endemic coronavirus RBD proteins were similar between the PCR-positive and PCR-negative individuals, suggesting that IgG against endemic coronavirus RBD regions was not associated with protection from infection. Moreover, we found no significant IgG antibody cross-reactivity between endemic coronaviruses and SARS-CoV-2 RBDs. Conclusions: Taken together, results suggest that anti-RBD antibodies induced by a previous infection with endemic HCoVs do not protect against acquisition of COVID-19 among exposed uninfected individuals.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Diverse amdoparvoviruses infection of farmed Asian badgers (Meles meles).

Amdoparvoviruses infect various carnivores, including mustelids, canids, skunks, and felids. Aleutian mink disease virus (AMDV) belongs to the prototypical species Amdoparvovirus carnivoran1. Here, we identified a novel amdoparvovirus in farmed Asian badgers (Meles meles), and we named this virus "Meles meles amdoparvovirus" (MMADV). A total of 146 clinical samples were collected from 134 individual badgers, and 30.6% (41/134) of the sampled badgers tested positive for amdoparvovirus by PCR. Viral DNA was detected in feces, blood, spleen, liver, lung, and adipose tissue from these animals. Viral sequences from eight samples were determined, five of which represented nearly full-length genome sequences (4,237-4,265 nt). Six serum samples tested positive by PCR, CIEP, and IAT, four of which had high antibody titers (> 512) against AMDV-G. Twenty-six of the 41 amdoparvovirus-positive badgers showed signs of illness, and necropsy revealed lesions in their organs. Sequence comparisons and phylogenetic analysis of the viral NS1 and VP2 genes of these badger amdoparvoviruses showed that their NS1 proteins shared 62.6%-88.8% sequence identity with known amdoparvoviruses, and they clustered phylogenetically into two related clades. The VP2 proteins shared 76.6%-97.2% identity and clustered into two clades, one of which included raccoon dog and arctic fox amdoparvovirus (RFAV), and the other of which did not include other known amdoparvoviruses. According to the NS1-protein-based criterion for parvovirus species demarcation, the MMADV isolate from farm YS should be classified as a member of a new species of the genus Amdoparvovirus. In summary, we have discovered a novel MMADV and other badger amdoparvoviruses that naturally infect Asian badgers and are possibly pathogenic in badgers.

Identification and characterization of three monoclonal antibodies targeting the SFTSV glycoprotein and displaying a broad spectrum recognition of SFTSV-related viruses.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne viral pathogen that causes severe fever with thrombocytopenia syndrome (SFTS). The disease was initially reported in central and eastern China, then later in Japan and South Korea, with a mortality rate of 13-30%. Currently, no vaccines or effective therapeutics are available for SFTS treatment. In this study, three monoclonal antibodies (mAbs) targeting the SFTSV envelope glycoprotein Gn were obtained using the hybridoma technique. Two mAbs recognized linear epitopes and did not neutralize SFTSV, while the mAb 40C10 can effectively neutralized SFTSV of different genotypes and also the SFTSV-related Guertu virus (GTV) and Heartland virus (HRTV) by targeting a spatial epitope of Gn. Additionally, the mAb 40C10 showed therapeutic effect in mice infected with different genotypes of SFTSV strains against death by preventing the development of lesions and by promoting virus clearance in tissues. The therapeutic effect could still be observed in mice infected with SFTSV which were administered with mAb 40C10 after infection even up to 4 days. These findings enhance our understanding of SFTSV immunogenicity and provide valuable information for designing detection methods and strategies targeting SFTSV antigens. The neutralizing mAb 40C10 possesses the potential to be further developed as a therapeutic monoclonal antibody against SFTSV and SFTSV-related viruses.

Evaluation of monkeypox- and vaccinia-virus neutralizing antibodies before and after smallpox vaccination: A sero-epidemiological study.

Since May 2022, several countries outside of Africa experienced multiple clusters of monkeypox virus (MPXV)-associated disease. In the present study, anti-MPXV and anti-vaccinia virus (VACV) neutralizing antibody responses were evaluated in two cohorts of subjects from the general Italian population (one half born before the WHO-recommended end of smallpox vaccination in 1980, the other half born after). Higher titers (either against MPXV or VACV) were observed in the cohort of individuals born before the interruption of VACV vaccination. An association between VACV and MPXV antibody levels was observed, suggesting that the smallpox vaccination may confer some degree of cross-protection against MPXV infection. Results from this study highlight low levels of immunity toward the assessed Orthopoxviruses, especially in young adults, advocating the introduction of a VACV- or MPXV-specific vaccine in case of resurgence of monkeypox disease outbreaks.

Smallpox vaccination campaigns resulted in age-associated population cross-immunity against monkeypox virus.

Increased human-to-human transmission of monkeypox virus (MPXV) is cause for concern, and antibodies directed against vaccinia virus (VACV) are known to confer cross-protection against Mpox. We used 430 serum samples derived from the Scottish patient population to investigate antibody-mediated cross-neutralization against MPXV. By combining electrochemiluminescence immunoassays with live-virus neutralization assays, we show that people born when smallpox vaccination was routinely offered in the United Kingdom have increased levels of antibodies that cross-neutralize MPXV. Our results suggest that age is a risk factor of Mpox infection, and people born after 1971 are at higher risk of infection upon exposure.

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.