A Simplified Sanger Sequencing Method for Detection of Relevant SARS-CoV-2 Variants.

Molecular surveillance of the new coronavirus through new genomic sequencing technologies revealed the circulation of important variants of SARS-CoV-2. Sanger sequencing has been useful in identifying important variants of SARS-CoV-2 without the need for whole-genome sequencing. A sequencing protocol was constructed to cover a region of 1000 base pairs, from a 1120 bp product generated after a two-step RT-PCR assay in samples positive for SARS-CoV-2. Consensus sequence construction and mutation identification were performed. Of all 103 samples sequenced, 69 contained relevant variants represented by 20 BA.1, 13 delta, 22 gamma, and 14 zeta, identified between June 2020 and February 2022. All sequences found were aligned with representative sequences of the variants. Using the Sanger sequencing methodology, we were able to develop a more accessible protocol to assist viral surveillance with a more accessible platform.

Chlamydia trachomatis Pgp3 Antibody Persists and Correlates with Self-Reported Infection and Behavioural Risks in a Blinded Cohort Study.

Chlamydia trachomatis (Ct) serological studies in populations could help monitor changes in lifetime cumulative risk of infection. We developed a double-antigen sandwich ELISA based on the Ct-specific Pgp3 antigen, then tested blind stored sera from over 800 participants in a New Zealand birth cohort from Dunedin at ages 26, 32 and 38. The double-antigen sandwich ELISA was more sensitive than our previously characterised indirect Pgp3 ELISA. Pgp3 antibody was detected more often in women compared to men and correlated with increasing numbers of sexual partners, self-reported Ct, and younger age at sexual debut in both women and men. At age 26, 24.1% (99/411) of women were Pgp3 seropositive, as were 79.5% (35/44) of those reporting Ct infection; Pgp3 antibody persisted to age 38 in 96.5% (83/86). In men at age 26, the figures were 10.7% (47/442) and 25.0% (6/24), respectively, with high (83.9%) antibody persistence to age 38. At age 38, among those Pgp3 seropositive, 63.3% of women and 83.1% of men had not reported Ct infection. Thus, Ct-specific Pgp3 antibody was detected in most women reporting Ct infection and correlated with risk of infection in those who did not, with most infections remaining undetected. As this antibody persisted for at least twelve years in 96% of these women, serology could be used to evaluate Ct prevention programmes among women.

C. trachomatis Pgp3 antibody prevalence in young women in England, 1993-2010.

Seroepidemiology of chlamydia can offer study opportunities and insights into cumulative risk of exposure that may contribute to monitoring the frequency of, and control of, genital chlamydia-the most commonly diagnosed STI in England. We undertook retrospective anonymous population-based cross-sectional surveys using an indirect IgG ELISA for chlamydia Pgp3 antibody. Sera from 4,732 women aged 17-24 years were tested. Samples were taken at 3-yearly intervals between 1993 and 2002, a period during which other data suggest chlamydia transmission may have been increasing, and from each year between 2007 and 2010. Seroprevalence increased in 17-24 year olds over time between 1993 and 2002. Between 2007 and 2010, age-standardised seroprevalence among 17-24 year olds decreased from 20% (95% CI: 17-23) to 15% (95%CI 12-17) (p = 0.0001). The biggest drop was among 20 to 21 year olds, where seroprevalence decreased from 21% in 2007 to 9% in 2010 (p = 0.002). These seroprevalence data reflect some known features of the epidemiology of chlamydia infection, and show that exposure to antibody-inducing chlamydia infection has declined in recent years. This decline was concurrent with increasing rates of screening for asymptomatic chlamydia. Serology should be explored further as a tool for evaluation of chlamydia control, including chlamydia screening programmes.

Human monoclonal antibodies to HIV-1 gp140 from mice bearing YAC-based human immunoglobulin transloci.

Mice carrying human immunoglobulin transloci were immunised with HIV-1 gp140 antigen to gain insight into the range and nature of human monoclonal antibodies (mAbs) that can be elicited from such humanised mice. Using five-feature mice that harbour YAC-based germline-configuration human IgM, Igκ and Igλ transloci in a mouse background disrupted for endogenous mouse IgH and Igκ expression, gp140-specific human IgM mAbs were readily elicited following serial immunisation. These mAbs were converted to human IgG1 format and were found to bind diverse epitopes within gp140, exhibiting high functional affinity for the antigen-typically in the nanomolar or sub-nanomolar range. The number of specific, stable hybridomas per mouse was, however, low (typically around five) with the hybridomas within individual mice often being clonally related. Nevertheless, different mice used B cell clones expressing varied V(D)J combinations, with affinity maturation through somatic hypermutation making a critical contribution. Thus, a wide range of distinct high-affinity mAbs can be obtained by immunising multiple animals. The results confirm the utility of the translocus-mouse approach and give insight into strategies for possible future improvement.

A novel strategy for inducing enhanced mucosal HIV-1 antibody responses in an anti-inflammatory environment.

Prophylactic vaccination against HIV-1 sexual transmission will probably require antibody elicitation at genital mucosal surfaces. However, HIV-1 envelope glycoprotein (Env)-based antigens are weakly immunogenic, particularly when applied mucosally. The polyanion PRO 2000 is safe for human vaginal application, and thus may represent a potential formulating agent for vaginal delivery of experimental vaccine immunogens. Based upon its biochemical properties, we hypothesized that PRO 2000 might enhance mucosal immunogenicity of HIV-1 envelope glycoprotein (Env)-based antigens, promoting local and systemic immune responses. Vaginal immunization with Env-PRO 2000 resulted in significantly increased titres of Env-specific mucosal IgA and IgG in mice and rabbits, respectively, compared to Env alone, revealing modest but significant mucosal adjuvant activity for PRO 2000. In vitro, PRO 2000 associated with Env, protecting the glycoprotein from proteolytic degradation in human vaginal lavage. Unexpectedly, PRO 2000 antagonized TLR4 activation, suppressing local production of inflammatory cytokines. Since inflammation-mediated recruitment of viral target cells is a major risk factor in HIV-1 transmission, the immune modulatory and anti-inflammatory activities of PRO 2000 combined with its intravaginal safety profile suggests promise as an HIV-1 mucosal vaccine formulating agent.

Preclinical evaluation of the immunogenicity of C-type HIV-1-based DNA and NYVAC vaccines in the Balb/C mouse model.

As part of a European initiative (EuroVacc), we report the design, construction, and immunogenicity of two HIV-1 vaccine candidates based on a clade C virus strain (CN54) representing the current major epidemic in Asia and parts of Africa. Open reading frames encoding an artificial 160-kDa GagPolNef (GPN) polyprotein and the external glycoprotein gp120 were fully RNA and codon optimized. A DNA vaccine (DNA-GPN and DNA-gp120, referred to as DNA-C), and a replication-deficient vaccinia virus encoding both reading frames (NYVAC-C), were assessed regarding immunogenicity in Balb/C mice. The intramuscular administration of both plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial T-cell responses against both antigens as well as Env-specific antibodies. Whereas low doses of NYVAC-C failed to induce specific CTL or antibodies, high doses generated cellular as well as humoral immune responses, but these did not reach the levels seen following DNA vaccination. The most potent immune responses were detectable using prime:boost protocols, regardless of whether DNA-C or NYVAC-C was used as the priming or boosting agent. These preclinical findings revealed the immunogenic response triggered by DNA-C and its enhancement by combining it with NYVAC-C, thus complementing the macaque preclinical and human phase I clinical studies of EuroVacc.

Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.