Increase in live infected cell number with drug and generation of a quasispecies are consequences of multiply HIV infected cells.

Doctoral Degree. University of KwaZulu-Natal, Durban.HIV may form reservoirs in anatomical compartments and evolve a quasispecies in order to survive under selective pressures such as antiretroviral drugs. Lymph nodes and lymphoid tissue - critical sites for reservoir formation - are environments conducive to cell-to-cell spread, an efficient mode of HIV transmission. Cell-to-cell spread can lead to multiple infections per cell which in turn profoundly changes how the virus responds to selective pressure. In this thesis, my goal was to understand the consequences of multiple infections per cell on how the infection responds to and evolves in the face of inhibitors. The specific aims were to: (1) model and experimentally examine the effect of attenuating cell-to-cell spread by using antiretrovirals (ARVs) on infected cell viability; (2) test whether a stable quasispecies can be formed and maintained by complementation– a process where virions derived from different HIV genotypes infecting the same cell share components; (3) test the feasibility of new single-cell RNA-Seq methodology that can be applied to quantify the frequency of multiply infected cells in vivo. These studies showed that: (1) partially attenuating infection involving multiple virions per cell with drug resulted in an increase in the number of live infected cells in both cell line and lymph nodes at suboptimal drug strengths. The increase in live infected cells was a result of fewer HIV DNA copies per cell, relative to no drug; (2) under the selective pressure of efavirenz (EFV), when drug-resistant and drug sensitive HIV co-infect the same cell during drug resistant evolution, complementation takes place, driving the formation and maintenance of a quasispecies; (3) Novel single-cell RNA-Seq approaches are feasible to quantify the number of cells that are multiply infected in vivo. Inhibiting mechanisms such as cell-to-cell spread may therefore reduce infection in the face of ARVs and limit viral diversity and hence the ability of HIV to evolve resistance

Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.African animal trypanosomosis or nagana is a disease in livestock caused by various species of protozoan parasites belonging to the genus Trypanosoma particularly T. congolense, T. vivax and T. b. brucei. Nagana is the most important constraint to livestock and mixed crop-livestock farming in tropical Africa. Trypanosomes undergo part of their developmental life in their insect vector, the tsetse fly and part in their mammalian host. Measures for eradicating the continent of the tsetse fly vector include insecticidal spraying, targeting and trapping. Vaccine development has been hampered by the generation of an inexhaustible collection of variant surface glycoproteins that trypanosomes possess and allow for evasion of the host immune system. Anti-disease vaccines aimed at reducing the symptoms of the disease rather than killing the parasite itself have been demonstrated as an alternative approach. Trypanotolerant cattle are able to protect themselves from the disease-associated symptoms. They are able to mount a better antibody response to the CATL-like cysteine peptidase, TcoCATL, compared to trypanosusceptible breeds. Bovine trypanosomosis, however, continues to be controlled primarily by trypanocidal compounds such as isometamidium chloride, homidium and diaminazene that have been developed more than 50 years ago and consequently drug resistance is widespread. Trypanosomal cysteine peptidases have also been proven to be effective targets for chemotherapeutics. TcrCATL, inhibited by the vinyl sulfone pseudopeptide inhibitor K11777, was effective in curing or alleviating T. cruzi infection in preclinical proof-of-concept studies and has now entered formal preclinical drug development investigation. Understanding enzymatic as well as structural characteristics of pathogenic peptidases is the first step towards successful control of the disease. To date no such characterisation of the major cysteine peptidases from T. vivax has been conducted. Although the major cysteine peptidase from T. vivax, TviCATL, has not been proven as a pathogenic factor yet, its high sequence identity with the pathogenic counterparts such as TcrCATL and TcoCATL hold much speculation for TviCATLs role in pathogenocity. In the present study, native TviCATL was isolated from T. vivax Y486, purified and characterised. TviCATL showed to have a general sensitivity to E-64 and cystatin and has a substrate specificity defined by the S2 pocket. TviCATL exhibited no activity towards the CATB-like substrate, Z-Arg-Arg-AMC but was able to hydrolyse Z-Phe-Arg-AMC, the CATL-like substrate. Leu was preferred in the P2 position and basic and non-bulky hydrophobic residues were accepted in the P1 and P3 positions respectively. Similar findings were reported for TcoCATL. The substrate specificity of TviCATL and TcoCATL does argue for a more restricted specificity compared to TcrCATL. This was based on the Glu333 in TcrCATL substituted with Leu333 in TviCATL and TcoCATL. In the case of TcrCATL, the Glu333 allows for the accommodation of Arg in the P2 position. Like other trypanosomal cysteine peptidases, TviCATL was inhibited by both chloromethyl ketones, Z-Gly-Leu-Phe-CMK and H-D-Val-Phe-Lys-CMK. Determining further structural and functional characteristics as well as whether TviCATL, like the T. congolense homolog, TcoCATL, acts as a pathogenic factor, would be important information to the designing of specific chemotherapeutic agents. To date, TcrCATL and TbrCATL (from T. b. rhodesiense) are the only trypanosomal CATL-like cysteine peptidases been crystallised and their tructures solved. This advantage has allowed for the directed design of synthetic peptidase inhibitors. The crystal structure of TcoCATL will be of major significance to the design of specific chemotherapeutic agents. Furtherrmore, understanding the dimeric conformation of TcoCATL is important for vaccine design as immune responses are likely to recognise the dimer specific epitopes. In the current study, the catalytic domain of TcoCATL and TviCATL, were recombinantly expressed in Pichia pastoris and purified to homogeneity. The T. congolense cysteine peptidase pyroglutamyl peptidase (PGP), also proven to be pathogenic in T. b. brucei, was recombinantly expressed in E. coli BL21 (DE3) cells and also purified to homogeneity. Purified cysteine peptidases along with previously purified TcoCATL dimerisation mutants, TcoCATL (H43W) and TcoCATL (K39F; E44P), possessing mutated residues involved in TcoCATL dimerisation, as well as the mutant proenzyme TcoCATL (C25A), were screened for crystallisation conditions using the Rigaku robotic crystallisation suite. One-dimensional needle-like crystals were found for TcoCATL (K39F; E44P). Optimisation of the TcoCATL (K39F; E44P) crystals were analysed for X-ray diffraction. The poor diffraction pattern prompted further optimisations for better crystal quality, which is presently underway. The crystal structure of TcoCATL, with some of the residues involved in dimerisation mutated, will be pivotal in understanding the dimerisation model. Furthermore, the information about the structure will be valuable for vaccine design and chemotherapeutics development

HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell

Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses

Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization

The emergence of Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections1. We investigated whether Omicron escapes antibody neutralization in South Africans vaccinated with Pfizer BNT162b2. We also investigated if Omicron requires the ACE2 receptor to infect cells. We isolated and sequence confirmed live Omicron virus from an infected person in South Africa and compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain, observing that Omicron still required ACE2 to infect. For neutralization, blood samples were taken soon after vaccination from participants who were vaccinated and previously infected or vaccinated with no evidence of previous infection. Neutralization of ancestral virus was much higher in infected and vaccinated versus vaccinated only participants but both groups showed a 22-fold escape from vaccine elicited neutralization by the Omicron variant. However, in the previously infected and vaccinated group, the level of residual neutralization of Omicron was similar to the level of neutralization of ancestral virus observed in the vaccination only group. These data support the notion that, provided high neutralization capacity is elicited by vaccination/boosting approaches, reasonable effectiveness against Omicron may be maintained

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements