immune evasion Flashcards
diversity in gp120 within and between clades?
20% within, 30% between
how many base changes per round of replication?
0.3
antigenic variation study
• Kimata et al, 1999, demonstrated that HIV variants increase in fitness as mutations accumulate over the course of an infection, demonstrating a form of co-evolution within the host
o Injected macaques intrarectally with SIV representing early, intermediate or late infections and found that ‘late-stage’ clones resulted in the greatest viral load (when measured with the Chiron branch DNA assay) mutations acquired by the virus maximise its capacity to persist within a specific host.
how much of gp120’s mass is glycans?
50%
transient exposure of env surfaces
• Demonstrated by Wei et al. 2003 Plasma samples of HIV infected individuals who had declined ART therapy were collected over the course of infection and neutralisation/infectivity assays were used to determine that the plasma contained neutralising Abs from approx. within weeks of the detection of HIV-specific Abs.
o Yet, the nAbs inhibitory activity resulted in the replacement of the entire viral population with escape mutants, containing mutations to the env gene, which primarily constituted changes to N-linked glycosylation
o [We thus propose an evolving ‘glycan shield’ that, within the quaternary confines of the trimer, acts as a steric hindrance preventing Nab from binding neighbouring epitopes, including those at V1/V2, V3, and the receptor binding surfaces]
o Shifting the shield to selectively accommodate receptor, but not antibody, alters the accessibility of neutralizing epitopes, and hence neutralization resistance
o Further, targeted mutations at 4 positions of the early HIV-nAb sensitive clones N-linked glycosylation sites to resemble escape mutants increased the IC50 of the clone by more than 100-fold, similar to those of highly resistant clones
pre-activation model of latency
o Swiggard et al, 2005, demonstrated in vitro spinoculation of resting CD4+ T cells could lead to the integration of HIV DNA and subsequent activation of these cells produced more HIV virus.
Flow cytometry of CD4+ population against activation markers CD69 & HLA-DR sorted CD4+ T cells into resting and activated populations
Resting cells were spinoculated with HIV virus (centrifuged for 2h to increase infection rate) and PCR analysis 3 days later found HIV DNA to have integrated in the CD4+ cells (0.13 provirus/cell).
Stimulating the resting cell population with IL-7 or anti CD-3/anti CD-28 beads caused 3-4.5% of the cell population to produce the Gag protein (measured by anti-Gag labelling)
post-activation model of latency
o Chavez et al (2015) developed a dual reporter HIV-virus (HIV Duo-Fluo), wherein eGFP replaced nef and an EF1α promoter causing mCherry expression was attached to the virus.
If only mCherry expressed, cell is latently infected, whilst if both mCherry and GFP expressed, cell is productively infected.
Primary CD4+ T cells from uninfected donor blood was stimulated using anti CD3/CD28 beads in presence of IL-2. As cells returned to their resting state, they were infected with HIV Duo-Fluo and then flow cytometry was used to see what type of infections had occurred.
Found that as cells returned to their resting state, the ratio of latent: productive infections increased, though the total number of infections fell, supporting the hypothesis that active T cells are the most permissive to HIV infection, but less susceptible to latency
o Critique – Kim et al, 2019, this model has been questioned, as there is a low constitutive expression of EF1α promotor in resting CD4+ T cells, meaning that latent CD4+ infection may be underestimated in resting cells according to recent work
molecular mechanisms of latency
o Tyagi et al, 2010, demonstrated that latently infected primary CD4+ memory cells proviruses are enriched in histone deacetylases and methylated histones. Further, restricted levels of p-TEFB (a viral promoter) in latently infected cells limited pro-viral transcription, despite high level of NF-κB being applied
viral reservoir established early
o Whitney et al. 2014 infected rhesus monkeys intrarectually with SIV and initiated ART therapy from 3 days post infection. Despite a lack of any viraemia, halting ART therapy after 24 weeks led to a viral rebound.
Critique – limited as SIV and a higher no. of viral copies intrarectally than natural infection
o Colby et al. 2018 supports these findings clinically. Study of 8 patients, where treatment had been initiated at the earliest stage of infection, where HIV 1 RNA is detectable without p24 antigen or seroconversion. Despite a median ART duration of 2.8 years, all patients experienced viral rebound upon interruption of ART.
cns symptoms
o ALLRT cohort (2011) studied 2636 individuals undergoing active ART, with undetectable viraemia. Individuals on ART regimes with higher CNS penetration had significantly greater increases in neuropsychological performance when adjusted for other variables (though this could be due to many effect)
lymph nodes as viral reservoirs
o Connick et al, 2014, demonstrated that the lymph node B cell follicle represents a potential location of a viral reservoir.
SIV infection of macaques intrarectally and harvested at different time points post infection (acute, chronic non-SAIDS and chronic SAIDS). Harvested lymphoid tissue and used FISH to demonstrate that vRNA is concentrated within the lymphoid tissue in the chronically infected macaque.
Chronically infected non-SAIDS animals had significantly more infected cells at follicular sites vs extrafollicular sites.
Hypothesised that this is due to SIV-specific CTL not being able to access the B cell follicles, which was confirmed by immunohistochemistry (fluorescent tagging). The over-expression of the extra-follicular retention molecule CCR7 on CTLs may explain why they did not enter the follicle.
Overall highlights a potential role of an immunologically priveliged site and highlights how CTLs are also immune evaded.
CD4+ HIV-specific
• Douek et al 2002. HIV may preferentially target HIV-specific T cells for infection.
o Used flow cytometry to separate HIV- and CMV-specific Tmemory ¬cells from infected individuals by antigen induced IFN-γ production.
o qpCR found that vDNA loads were 3.7x higher in HIV-specific CD4+ cells vs CMV-specific cells, suggesting a preferential infection.
o allows more CD4+ cells to be infected (expanding the HIV-population), without significantly compromising the immune response to other pathogens (which is crucial for host survival and, thus, HIV persistence)
o Critique – CMV specific T cells may be abnormally resistant, and a larger study ought to be carried out with analyses to account for confounders.
nef
• Swigut et al. 2004 infected monkeys with Nef-mutant SIV, where the Nef was non-functional for the downregulation of MHC Class I (though other effects cannot be accounted for). Monkeys infected with the Nef-mutant SIV demonstrated significantly greater CD8+ T cell responses 1-4 months post infection (measured by Gag tetramer staining) compared to wild-type infections, highlighting the role that Nef plays in limiting the host immune system.
evading immunodominant epitopes
o One such immunodominant epitope in the CD8+ response to HIV is gag p24 263-272 in individuals with HLA B27 haplotype.
o Ammaranond et al. conducted a retrospective study on an Australian cohort of HLAB*27+ HIV+ long term non-progressors. Out of the 19 patients identified, sequencing of plasma samples taken previously found that 7 of them carried a mutation at position 264 of gag p24 region. Dividing the group into WT and escape mutants of codon 264, it was found that whilst at study entry there was no difference in viral load between groups, at follow-up (>10 years later) the escape mutant group had a significantly higher viral load than WT, which couldn’t be explained by other common factors affecting viral load.
evading tetherin
• Neil et al (2008) = identified tetherin through comparative transcriptomics
- looked at levels of mRNA from different cell types by microarray
- looked for genes with constitutive expression in restrictive cells (e.g. HeLa), low expression in cell lines where Vpu is dispensable (e.g. HOS)
• identified tetherin = expression > 20-fold greater in HeLa than HOS
• also used EM to look at the appearance of surface of infected cells expressing tetherin or not
- cells not expressing tetherin = few virus particles visible as viruses have all budded off
- cells expressing tetherin = lots of virus particles tethered to cell surface, as they cannot bud off properly
