Assessing TMV as an immunogenic particle : the expression of HIV-1 subtype C V3 loop neutralizing antibody epitopes on the surface of TMV virions

Includes bibliographical references (leaves 105-120).In an attempt to establish a plant-based Human immunodeficiency virus-1 (HIV-1) subtype C neutralizing antibody stimulating vaccine, a Tobacco mosaic virus (TMV) derived vector was used to express recognized HIV neutralizing antibody epitopes. These epitopes were expressed on the surface of the TMV coat protein, which served as an ideal means of antigen display. This model antigen display system was capable of assembling into multivalent, highly repetitive structures thereby displaying many copies of the attached epitope in the assembled virion. Three TMV-based vectors were acquired, which were essentially identical, differing only in the position at which they could accommodate a foreign protein fusion. These vectors allowed the display of a foreign peptide at the N-terminus, Cterminus or 60S-loop regions of the TMV coat protein. all of which protrude on the surface of the assembled virion. The HIV V3 loop is recognized as the principal neutralizing domain, and was the neutralizing epitopes displayed by the vectors. The epitope sequences used were derived from a cohort of infected individuals in Durban, South Africa, who displayed broad cross-neutralizing V3-specific activity towards heterologous viral strains. The recombinant viral vectors were shown to efficiently infect the host Nicotiana benthamiana plants and assemble into multivalent recombinant virion structures as observed by transmission electron microscopy. However, the level of coat protein expression was significantly dependent on the position of the coat protein fusion as either the levels of V3 epitope expressed or TMV coat protein was found to vary between the different vector types, confirmed by means of immunoblotting and enzyme-linked immunosorbent assays. Immunogenicity analysis using a guinea pig model was used to assess the ability of the recombinant vectors to firstly establish a V3-specific immune response, and secondly to stimulate a virus-specific neutralization antibody response. As a result of time constraints only the C-terminal coat protein fusions were assessed in the guinea pig model. Inoculated guinea pigs displayed distinct and gradually increasing V3-specific immune responses after 2 boosts. Serum samples that displayed the strongest V3 peptide responses were then analyzed for their ability to neutralize HIV infection in HIV pseudovirion neutralization assays. Results for selected serum samples showed no HIV neutralizing activity above what could be recognized as background activity. Thus the candidate vaccine, although establishing a path for the assembly of a multivalent vaccine, failed in its attempt to stimulate a neutralizing antibody response. This study has nevertheless paved a direct path to the development of variations of this type of vaccine possibly using different and perhaps more effective epitopes for candidate vaccine purposes

RNA transmission and expression from inert HIV candidate vaccine virus-like-particles

Includes abstract.Includes bibliographical references (leaves 136-158).HIV-1 Gag virus-like-particles (VLPs) produced in various expression systems are potent stimulators of both cellular and humoral immune responses in animal models. The encapsidation of large concentrations of random cellular RNA species is known to accompany the assembly of HIV virus particles. This RNA plays a crucial role by serving as a molecular scaffold for the assembly of Gag structural proteins into particles. Non-pseudotyped VLPs that do not present any HIV envelope glycoproteins are regarded as inert particles as they contain no replicative nucleic acid and are presumed to be unable to deliver encapsidated RNA for expression in inoculated individuals. Live virus cellular entry studies have shown that non-pseudotyped Gag particles are destined for degradation in acidified vesicles subsequent to receptor independent cellular entry. In addition to host cell RNA incorporation, Gag VLPs produced in insect cell-based, baculovirus expression systems have been observed to incorporate the baculovirus-derived Gp64 envelope glycoprotein. Gp64 has been shown to be efficient at enabling the delivery and expression of genes from recombinant baculoviruses and other Gp64 pseudotyped live viruses in mammalian cell lines both in vivo and in vitro. This study, therefore, set out to establish for the first time whether inert, baculovirus-derived (Gp64 pseudotyped) Gag VLPs could mediate delivery and expression of randomly encapsidated RNAs in mammalian cell lines

Phylogenetic exploration of nosocomial transmission chains of 2009 influenza A/H1N1 among children admitted at Red Cross War Memorial Children's Hospital, Cape Town, South Africa in 2011

Traditional modes of investigating influenza nosocomial transmission have entailed a combination of confirmatory molecular diagnostic testing and epidemiological investigation. Common hospital-acquired infections like influenza require a discerning ability to distinguish between viral isolates to accurately identify patient transmission chains. We assessed whether influenza hemagglutinin sequence phylogenies can be used to enrich epidemiological data when investigating the extent of nosocomial transmission over a four-month period within a paediatric Hospital in Cape Town South Africa. Possible transmission chains/channels were initially determined through basic patient admission data combined with Maximum likelihood and time-scaled Bayesian phylogenetic analyses. These analyses suggested that most instances of potential hospital-acquired infections resulted from multiple introductions of Influenza A into the hospital, which included instances where virus hemagglutinin sequences were identical between different patients. Furthermore, a general inability to establish epidemiological transmission linkage of patients/viral isolates implied that identified isolates could have originated from asymptomatic hospital patients, visitors or hospital staff. In contrast, a traditional epidemiological investigation that used no viral phylogenetic analyses, based on patient co-admission into specific wards during a particular time-frame, suggested that multiple hospital acquired infection instances may have stemmed from a limited number of identifiable index viral isolates/patients. This traditional epidemiological analysis by itself could incorrectly suggest linkage between unrelated cases, underestimate the number of unique infections and may overlook the possible diffuse nature of hospital transmission, which was suggested by sequencing data to be caused by multiple unique introductions of influenza A isolates into individual hospital wards. We have demonstrated a functional role for viral sequence data in nosocomial transmission investigation through its ability to enrich traditional, non-molecular observational epidemiological investigation by teasing out possible transmission pathways and working toward more accurately enumerating the number of possible transmission events

Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

Background: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods. HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (∼1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/g Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice. © 2011 Valley-Omar et al; licensee BioMed Central Ltd

Human Parainfluenza Virus (HPIV) Detection in Hospitalized Children with Acute Respiratory Tract Infection in the Western Cape, South Africa during 2014–2022 Reveals a Shift in Dominance of HPIV 3 and 4 Infections

The epidemiology of human parainfluenza viruses (HPIV), particularly its role as a cause of acute respiratory infection (ARI) in infants, has not been formally studied in South Africa. We evaluated HPIV prevalence in diagnostic samples from hospitalized children from public sector hospitals in the Western Cape between 2014 and 2022. HPIV infection was detected in 2–10% of patients, with the majority of infections detected in children less than 1 year of age. Prior to 2020, HPIV 4 (40%) and HPIV 3 (34%) were the most prevalent types, with seasonal peaks in late winter/spring for HPIV 3 and autumn/winter for HPIV 4. HPIV 4A and 4B co-circulated during the seasonal activity between 2014 and 2017. Pandemic restrictions in 2020 had a profound effect on HPIV circulation and the rebound was dominated by waves of HPIV 3, accounting for 66% of detections and a sustained decline in the circulation of HPIV 1, 2 and 4. An immunity gap could account for the surge in HPIV 3 infections, but the decline in prior HPIV 4 dominance is unexplained and requires further study

Human respiratory syncytial virus diversity and epidemiology among patients hospitalized with severe respiratory illness in South Africa, 2012-2015

BACKGROUND: We aimed to describe the prevalence of human respiratory syncytial virus (HRSV) and evaluate associations between HRSV subgroups and/or genotypes and epidemiologic characteristics and clinical outcomes in patients hospitalized with severe respiratory illness (SRI). METHODS: Between January 2012 and December 2015, we enrolled patients of all ages admitted to two South African hospitals with SRI in prospective hospital-based syndromic surveillance. We collected respiratory specimens and clinical and epidemiological data. Unconditional random effect multivariable logistic regression was used to assess factors associated with HRSV infection. RESULTS: HRSV was detected in 11.2% (772/6908) of enrolled patients of which 47.0% (363/772) were under the age of 6 months. There were no differences in clinical outcomes of HRSV subgroup A-infected patients compared with HRSV subgroup B-infected patients but among patients aged <5 years, children with HRSV subgroup A were more likely be coinfected with Streptococcus pneumoniae (23/208, 11.0% vs. 2/90, 2.0%; adjusted odds ratio 5.7). No significant associations of HRSV A genotypes NA1 and ON1 with specific clinical outcomes were observed. CONCLUSION: While HRSV subgroup and genotype dominance shifted between seasons, we showed similar genotype diversity as noted worldwide. We found no association between clinical outcomes and HRSV subgroups or genotypes.Centers for Disease Control and Prevention,https://onlinelibrary.wiley.com/journal/17502659Medical Virolog

Household transmission of seasonal influenza from HIV-infected and HIV-uninfected individuals in South Africa, 2013-2014

BACKGROUND : We estimated the household secondary infection risk (SIR) and serial interval (SI) for influenza transmission from HIV-infected and HIV-uninfected index cases. METHODS : Index cases were the first symptomatic person in a household with influenza-like illness, testing influenza positive on real-time reverse transcription polymerase chain reaction (rRT-PCR). Nasopharyngeal swabs collected from household contacts every 4 days were tested by rRT-PCR. Factors associated with SIR were evaluated using logistic regression. RESULTS : We enrolled 28 HIV-infected and 57 HIV-uninfected index cases. On multivariable analysis, HIV-infected index cases were less likely to transmit influenza to household contacts (odds ratio [OR] 0.2; 95% confidence interval [CI], 0.1–0.6; SIR 16%, 18/113 vs 27%, 59/220). Factors associated with increased SIR included index age group 1–4 years (OR 3.6; 95% CI, 1.2–11.3) and 25–44 years (OR 8.0; 95% CI, 1.8–36.7), and contact age group 1–4 years (OR 3.5; 95% CI, 1.2–10.3) compared to 5–14 years, and sleeping with index case (OR 2.7; 95% CI, 1.3–5.5). HIV infection of index case was not associated with SI. CONCLUSIONS : HIV-infection was not associated with SI. Increased infectiousness of HIV-infected individuals is likely not an important driver of community influenza transmission.The National Institute for Communicable Diseases of the National Health Laboratory Service and the US Centers for Disease Control and Prevention [co-operative agreement number: 5U51IP000155.https://academic.oup.com/jid2020-05-15hj2019Medical Virolog

CAPRISA 004 tenofovir microbicide trial: no impact of tenofovir gel on the HIV transmission bottleneck.

Alterations of the genital mucosal barrier may influence the number of viruses transmitted from a human immunodeficiency virus–infected source host to the newly infected individual. We used heteroduplex tracking assay and single-genome sequencing to investigate the effect of a tenofovir-based microbicide gel on the transmission bottleneck in women who seroconverted during the CAPRISA 004 microbicide trial. Seventy-seven percent (17 of 22; 95% confidence interval [CI], 56%–90%) of women in the tenofovir gel arm were infected with a single virus compared with 92% (13 of 14; 95% CI, 67%–>99%) in the placebo arm (P = .37). Tenofovir gel had no discernable impact on the transmission bottleneck

Clinical characterisation and phylogeny of respiratory syncytial virus infection in hospitalised children at Red Cross War Memorial Children’s Hospital, Cape Town

Abstract Background Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young children in both the community and hospital setting. Methods The clinical presentation, patient and phylogenetic characteristicsof laboratory-confirmed cases of RSV, as well as risk factors for nosocomial infectionat Red Cross War Memorial Children’s Hospital in Cape Town were analysed. A multiplex PCR assay that detects 7 respiratory viruses was used to identify RSV nucleic acid on respiratory specimens. Results A total of 226 children were studied, ages ranging between 1 week and 92.5 months (median: 2.8 months, IQR: 1.3–6.3 months) and 51.8 % were males. The median duration of symptoms prior to diagnosis was 2 days (IQR: 1–4 days). Nosocomial infections wereidentified in 22 (9.7 %) children. There were pre-existing medical conditions in 113 (50.0 %) excluding HIV, most commonly prematurity (n = 58, 50.0 %) and congenital heart disease (n = 34, 29.3 %). The commonest presenting symptoms were cough (196, 86.7 %), difficulty in breathing (115, 50.9 %) and fever (91, 41.6 %).A case fatality rate of 0.9 % was recorded. RSV group A predominated (n = 181, 80.1 %) while group B accounted for only 45 (19.9 %) of the infections. The prevalent genotypes were NA1 (n = 127,70.1 %), ON1 (n = 45,24.9 %) and NA2 (n = 9,5.0 %) for group A while the only circulating RSV B genotype was BA4. There was no significant difference in the genotype distribution between the nosocomial and community-acquired RSV infections. Age ≥ 6 months was independently associated with nosocomial infection. Conclusions A large percentage of children with RSV infection had pre-existing conditions. Approximately one tenth of the infections were nosocomial with age 6 months or older being a risk factor. Though both RSV groups co-circulated during the season, group A was predominant and included the novel ON1 genotype. Continued surveillance is necessary to identify prevalent and newly emerging genotypes ahead of vaccine development and efficacy studies