Expression of HIV-1 subtype C nef in E. coli and Nicotiana benthamiana : development of plant-based vaccines for HIV

Bibliography: leaves 126-145.Expression of the nefgene from HIV-1 subtype C in Nicotiana benthamiana was carried out using a TMV -based vector with the aim of developing a plant-based candidate vaccine for HIV-1. The nef gene of the DU151 isolate of mV-l subtype C taken from a recently seroconverted individual was amplified by PCR with a deletion of 59 amino acids from the cytotoxic N-terminal. The amplified gene was inserted into a bacterial expression vector pProEXHTb for rapid expression of Nef protein, which was used as a diagnostic tool in the development of an indirect ELISA assay for detection of Nef in Nicotiana benthamiana. An indirect ELISA assay was developed using a commercially available polyclonal anti Nef antiserum raised in sheep. The role of codon optimization in expression of Nef in benthamiana was investigated. A synthetic nef gene was constructed based on the codon usage of benthamiana. The plant codon optimized gene and the wild type nef genes were inserted into the TMV -based vector pBSG1057. RNA transcripts from both constructs were used to infect young benthamiana plants. Expression of nefmRNA was confirmed by RT -PCR analysis of total RNA extracted from plants inoculated with respective constructs. The Nef protein was expressed at low levels which were detectable by ELISA. Nef was detectable by Western blot after concentration of plant extract using a membrane filter device. Quantitative analysis of Nef expression in plants was done by western blot on concentrated plant extract from three separate infections. Codon optimization of the nef gene improved the expression of Nef by a factor of about two

Understanding the interplay between HIV-1 diversity, humoral immune responses and viral fitness

Includes bibliographical references.HIV-1 antibody dependent cell cytotoxicity (ADCC) and neutralizing antibody (nAb) responses are both thought to be important responses to elicit through vaccination. This thesis characterises neutralizing response in two cohorts in Africa, and in a detailed study of one individual, elucidates the interplay between ADCC and nAb responses in early infection, and the impact of humoral escape on viral fitness. It remains an open question whether different geographically distinct population groups vary in their neutralization responses to HIV-1. We compared neutralizing antibody responses in two African cohorts and found 35% of the Tanzanians in the HIV Superinfection Study (HISIS) cohort had neutralization breath (neutralized >50% of panel viruses) at two years post infection compared to only 9% in the Centres for AIDS Program of Research in South Africa (CAPRISA) cohort. Cumulative viral loads between 3 and 12 months post infection were strongly associated with neutralization breadth (p<0.001), and were higher in the Tanzanian cohort (p=0.046). No association was found between breadth and dual infection, subtype or features of the envelope. One elite neutralizer was identified in the HISIS cohort with responses targeting the CD4 binding site. While neutralizing antibodies are considered central for protection from infection, ADCC activity correlated with reduced risk of HIV-1 acquisition in the RV144 trial. There is limited understanding of the overlapping ADCC and neutralizing antibody functions in early infection. We investigated the kinetics and targets of both responses in one individual from CAPRISA. ADCC responses were detected 4 weeks post infection, with nAbs responses emerging at 7 weeks post infection. We identified five neutralization escape patterns in the V4 region of the envelope by 11 weeks post-infection. Four of these also conferred ADCC escape; however the fifth neutralization escape variant resulted in increased sensitivity to ADCC. This variant was eliminated in vivo by 29 weeks post infection. Finally, we studied the effect of neutralizing and ADCC antibody escape mutations on the virus' ability to mediate fusion, infectivity and replicative fitness. Envelopes bearing immune escape adaptations had lower cell-cell fusion ability compared to the T/F virus. The mutations also resulted in reduced infectivity of infectious molecular clone virus stocks. However, only the largest deletion in the V4 caused reduced growth in peripheral blood mononuclear cells (PBMC). In conclusion, the study finds that neutralizing antibody responses are influenced by community viral loads. The study defined the first ADCC epitope reported in the V4 region, and describes overlapping targets for ADCC and neutralizing antibodies. Where neutralization escape resulted in increased ADCC sensitivity, this was a dead end escape pathway. Finally we find that early V4 escape from both ADCC and nAb responses had a fitness impact on the virus. We thus demonstrate a mechanism through which ADCC and neutralizing antibodies can synergistically influence viral evolution and potentially produce a protective immune response

Conserved positive selection signals in gp41 across multiple subtypes and difference in selection signals detectable in gp41 sequences sampled during acute and chronic HIV-1 subtype C infection

<p>Abstract</p> <p>Background</p> <p>The high diversity of HIV variants driving the global AIDS epidemic has caused many to doubt whether an effective vaccine against the virus is possible. However, by identifying the selective forces that are driving the ongoing diversification of HIV and characterising their genetic consequences, it may be possible to design vaccines that pre-empt some of the virus' more common evasion tactics. One component of such vaccines might be the envelope protein, gp41. Besides being targeted by both the humoral and cellular arms of the immune system this protein mediates fusion between viral and target cell membranes and is likely to be a primary determinant of HIV transmissibility.</p> <p>Results</p> <p>Using recombination aware analysis tools we compared site specific signals of selection in gp41 sequences from different HIV-1 M subtypes and circulating recombinant forms and identified twelve sites evolving under positive selection across multiple major HIV-1 lineages. To identify evidence of selection operating during transmission our analysis included two matched datasets sampled from patients with acute or chronic subtype C infections. We identified six gp41 sites apparently evolving under different selection pressures during acute and chronic HIV-1 infections. These sites mostly fell within functional gp41 domains, with one site located within the epitope recognised by the broadly neutralizing antibody, 4E10.</p> <p>Conclusion</p> <p>Whereas these six sites are potentially determinants of fitness and are therefore good candidate targets for subtype-C specific vaccines, the twelve sites evolving under diversifying selection across multiple subtypes might make good candidate targets for broadly protective vaccines.</p

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

Limited Neutralizing Antibody Specificities Drive Neutralization Escape in Early HIV-1 Subtype C Infection

We previously showed that HIV-1 subtype C viruses elicit potent but highly type-specific neutralizing antibodies (nAb) within the first year of infection. In order to determine the specificity and evolution of these autologous nAbs, we examined neutralization escape in four individuals whose responses against the earliest envelope differed in magnitude and potency. Neutralization escape occurred in all participants, with later viruses showing decreased sensitivity to contemporaneous sera, although they retained sensitivity to new nAb responses. Early nAb responses were very restricted, occurring sequentially and targeting only two regions of the envelope. In V1V2, limited amino acid changes often involving indels or glycans, mediated partial or complete escape, with nAbs targeting the V1V2 region directly in 2 cases. The alpha-2 helix of C3 was also a nAb target, with neutralization escape associated with changes to positively charged residues. In one individual, relatively high titers of anti-C3 nAbs were required to drive genetic escape, taking up to 7 weeks for the resistant variant to predominate. Thereafter titers waned but were still measurable. Development of this single anti-C3 nAb specificity was associated with a 7-fold drop in HIV-1 viral load and a 4-fold rebound as the escape mutation emerged. Overall, our data suggest the development of a very limited number of neutralizing antibody specificities during the early stages of HIV-1 subtype C infection, with temporal fluctuations in specificities as escape occurs. While the mechanism of neutralization escape appears to vary between individuals, the involvement of limited regions suggests there might be common vulnerabilities in the HIV-1 subtype C transmitted envelope

Features of recently transmitted HIV-1 clade C viruses that impact antibody recognition : implications for active and passive immunization.

CAPRISA, 2016.Abstract available in PDF file

Medical cannabis and cannabidiol: A new harvest for Malawi

In February 2020 parliament passed the Cannabis Regulation Bill (2020) which regulates the cultivation and production of industrial hemp and medical cannabis. The country will only fully benefit from this development if the medical and scientific community can take the lead in enabling the country to exploit the plant’s potential to help address some of our economic and public health challenges. This special communication provides some basic information on cannabis and discusses its history and medical uses. Cannabidiol (CBD) has emerged as one of the most important cannabis-derived phytochemicals and has formed the basis for the growth of the medical cannabis industry. The scientific data on the mechanisms of the effects of CBD on the human neuroendocrine-immune network is reviewed and the first effective cannabis-based FDA-approved treatment for epilepsy discussed. Some clinical research that is being done on the antipsychotic and neuroprotective properties of CBD is also reviewed. A case is made for the potential of CBD as a neuroprotective adjunctive therapy for the prevention of neuropsychological sequelae associated with complicated malaria. The safety profile of CBD is reviewed and finally, the potential importance of the re-medicalization of cannabis-based therapies for the broader field of phytomedicine is pointed ou

The emergence of SARS-CoV-2 variants that may affect COVID-19 vaccine efficacy - a systematic review

Background and Aim of the study: Recent developments in the fight against the COVID-19 pandemic, have demonstrated that SARS-CoV-2 is rapidly adapting to its new human host through viral evolution. The independent emergence of new SARS-CoV-2 variants globally is now a major factor to be taken into consideration in the development and deployment of vaccines and therapeutics. This systematic review highlights a growing body of literature examining the possible impact of these variants on the global COVID-19 vaccine development and vaccination programs underway. Method: Data were gathered from multiple sources such as google scholar and databases such as PubMed, Science Direct, and Research Gate with search terms such as “SARS-CoV-2 variant”. The Snowball method was used to track down other related articles. Eligible literature was only those in line with the objective of the review, and published from January 2020, up to January 2021. Result: The reviewed literature highlights the primary impact of the independent variants such as increased transmissibility relative to the ancestral strain, prolonged viral shedding and delayed viral clearance, and reduced neutralizing capacities of antibodies elicited by ancestral strains. Conclusion: The emergence of the new strain with increased fitness in terms of transmissibility and immune escape poses a serious challenge to the successful deployment of one of the strongest public health measures available at our disposal in the fight against COVID-19. Increasing global capacity for genomic surveillance and adaptability in the recalibration of vaccine and therapeutic programs will be important attributes to develop for the successful management of this major global health crisis

'Not all fevers are malaria': a mixed methods study of non-malarial fever management in rural southern Malawi.

IntroductionWith the ability to diagnose malaria with rapid diagnostic tests (mRDT), interest in improving diagnostics for non-malarial fevers has increased. Understanding how health providers diagnose and treat fevers is important for identifying additional tools to improve outcomes and reduce unnecessary antibiotic prescribing, particularly in areas where access to laboratory diagnostics is limited. This study aimed to understand rural health providers' practice patterns, both quantitatively and qualitatively, and influences on diagnostic and treatment decision-making.MethodsA mixed-methods study was conducted in Mulanje and Phalombe districts in southern Malawi. Retrospective data on diagnoses and treatments of febrile illness from seven mobile clinic logbooks were collected for a 2-month period in both the dry and wet seasons. Mobile health clinics visited remote villages in southern Malawi once every 7 days. Records from all patients with a recorded axillary temperature of 37.5ºC or higher or reported history of fever within 48 hours, and a negative mRDT, were included in the analysis. Key informant interviews were conducted with 31 mobile clinic health workers who triage, diagnose, and treat patients as well as dispense medication.ResultsIn total, 30 672 febrile patients were seen during the study period. Of those, 9924 (32%) tested negative for malaria by mRDT. Acute respiratory infection was the most common diagnosis for mRDT-negative patients (44.6%), and this number increased in the rainy season as compared to the dry season (odds ratio=2.18, 95% confidence interval=2.01-2.36). Over half (60%) of mRDT-negative patients received antibiotics as a treatment. Almost all the health providers in this study reported limited training in non-malarial fever management, despite the fact that roughly 30% of all patients with fever seen at the mobile clinics tested negative by mRDT. Without diagnostic tools beyond mRDTs, providers relied heavily on patient history to guide treatment decisions.ConclusionAdditional simple-to-use diagnostic tests as well as additional training in patient examination and clinical assessment are needed in rural settings where health providers risk over-prescribing antibiotics or missing a potentially dangerous infection in febrile patients who test negative for malaria