HIV life cycle and pathogenesis Flashcards
importance of CCR5 study
- The importance of the CCR5 coreceptor interaction in HIV pathogenesis is demonstrated by the fact that CCR5 mutations which result in reduced or abolished CCR5 expression provide protection from HIV infection.
Liu et al (1996) identified the protective effect of CCR5 mutations. They isolated CD4+ T cells from HIV-resistant patients, and these were unable to be infected by HIV in vitro, and they subsequently identified a homozygous defect (a 32-base pair deletion) in CKR-5, the gene encoding the CCR5 coreceptor.
Patients homozygous for this mutation are completely HIV-resistant, whilst heterozygous individuals have a slower development of the disease.
importance of protease activity
Kohl et al (1988) mutated the viral protease with a point mutation at residue 25, exchanging aspartic acid for asparagine, so that it was no longer functional. This resulted in a loss of formation of p24 in E. Coli cells, and the HIV viruses produced were unable to infect MT-4 lymphoid cells (a T cell line highly sensitive to HIV-1). This indicates the importance of the protease activity in virulence of HIV.
type I IFN study
Sandler et al. (2014) – looked at the effects of IFN on early and later SIV infection of macaques…
• IFN-1ant (antagonist; blocks type I IFN receptors and thus any type I IFN response) treatment (4 weeks of treatment prior to infection) significantly increased viral load and increased subsequent mortality rates when compared to controls who received carrier solution only
o i.e. knocking out the IFN response -> higher viral load + mortality -> suggests type I IFN plays an important protective role early on, reducing viral load.
• Pre-treatment with Type I IFN resulted in increased resistance of macaques to SIV infection (vs. placebo)
o Placebo group were all infected with 1 high does SIV challenge,
o In the group pre-treated with type I IFN, it took up to 5 challenges for all to become infected.
o Conclusion 🡪 type I IFN at v. early time point is beneficial vs. infection.
• HOWEVER – longer term IFN treatment after infection increased CD4+ T cell loss (again vs. placebo),
o Hypothesised to be a result of increased CD4+ T cell activation (since type I IFN is a pro-inflammatory cytokine and will lead to recruitment and activation of at least subpopulations of CD4+ T cells).
• OVERALL CONCLUSION:
o At v. early time point type I IFN is protective – slowing down/ aborting infection, HOWEVER, chronic elevation of type I IFN has a negative effect due to increased T cell activation and thus increased viral replication.
This illustrates the dichotomy of effect of inflammation in HIV-1 whilst immune activation is vital to attempt to supress HIV-1 replication, it can also facilitate its spread and subsequent replication.
when is AIDS declared?
CD4+ T cell counts below 200/mm^3
immune activation is predictive of AIDS progression
Immune activation has been found to be predictive of progression to AIDS
Giorgi et al (1999)
- measured cd38 activation marker expression on CD4+ cells of AIDS patients
(incubated cells with fluorescently tagged mAbs = measured median fluorescence intensity)
- CD38 expression strongly correlated with rate of disease progression whereas VIRAL LOAD DID NOT
- Higher numbers of CD38 molecules on CD4+ cells in group that progressed to AIDS more quickly, despite similar viral loads
This is corroborated by difference seen in elite controllers and rapid progressors, low level non-specific immune response vs high non-specific immune response
AIDs signs and symptoms
opportunistic infections
e.g. encephalitis and meningitis, pneumonia, TB, inflammatory gastrointestinal conditions worsened by co-infections, tumours (e.g. Kaposi’s sarcoma caused by HHV8), cognitive impairment
mucosal stromal fibroblasts study
• Recent study by Neidleman et al. 2017, illustrated the ability of mucosal stromal fibroblasts to enhance HIV infection of CD4+ T cells, First, they isolated endometrial stromal fibroblasts (eSFs) and co-cultured them by HIV and CD4 T cells, 6-100 fold increases of CD4 T cell infection rates were seen in their presence, when different donor eSFs and T cells were used. The enhancement of ESF to infectivity when compared to their absence increased as HIV load decreased. To confirm that the enhancement of CD4 infection was due to trans-infection and not production of more virions by eSFs same experiments were done with replication incompetent single-round virus, the same results were observed. The only shortcoming of the study was it’s inability to elucidate the molecular mechanism of HIV binding to eSF although they did rule out CD169 and DC-sign. Significant because stromal fibroblasts abundant in mucosal tissue directly below epithelial cells, therefore likely to play role in transmission and initial infection, could explain why viral load of 1 can cause HIV infection.
LFA-1 in virological synapses
• Cell-cell junction strengthened by ICAM-1/LFA-1 interaction, recruited on GP120-CD4 binding. Starling et al. 2016 found that it is LFA-1 engagement with the infected cell that induces polarisation causing movement of the microtubule organising centre and intracellular env to the virological synapse. This was illustrated by polarisation being achieved using anti-LFA-1 β2 antibody-coupled beads and a reduction in polarisation seen when LFA-1 negative cells were used
other mechanisms of cell-cell transfer
- Some evidence to suggest phagocytosis by macrophages of dying HIV infected CD4 cells causes macrophage infection. Baxter et al. 2014 showed through multispectral flow cytometry that monocyte-derived macrophages preferentially engulfed HIV infected cells. And found that post merging HIV vDNA was present within the macrophages. The full mechanism for this process is little understood however it appears to occur independently of synapse formation.
- Other mechanisms include syncytium formation where the fusion of membranes occurs between an infected cell and a target cell through GP120-CD4 interaction. How this contributes to pathogenesis is unclear, however recent evidence in humanised mice and 3D cultures suggest that multi-nucleated cells resulting from HIV-mediated cell-fusion are viable.
- Long distance cell-cell connections via nanotubules have also been described. Some studies suggest this allows transfer of extracellular viral particles but other work done in macrophages suggests transfer occurs through endosomes containing intracellular virus particles (Kadiu and Gendelman 2011)
- Transcytosis in mucosal epithelial cells is thought to play an in important role in initial HIV transmission, here HIV is internalised into vesicles at the apical surface and transported to the basal layer where it is released and exposed to target cells. Kinlock et al. 2014 illustrated that HIV-1 transcytosis occurs through the tubulation-dependent endocytic recycling pathway. This was illustrated by addition of BEL, an inhibitor of endosome tubulation and hence able to inhibit trafficking from the recycling endosome, reducing HIV-1 release and hence transcytosis.
effect of CCT on ART
- The effect of HIV cell-cell transmission on ART efficacy is a much debated topic. Some studies have indicated reduced efficacy whilst others have argued the contrary.
- Some nucleoside analogue reverse transcriptase inhibitors have been shown to be less effective against cell-cell transmission Agosto et al. 2014 showed multiple orders of magnitude increase in the IC90 of NRTIs Tenofovir and AZT in a cell-cell transmission model using Jurkat cells (immortalised line of human CD4 T cells) compared to a cell-free model.
- In contrast other ART drugs investigated by Agosto et al. 2014 were shown to retain efficacy across cell-free and cell-cell transmission models and found that combination therapies of NRTIs found to have reduced efficacy against cell-to-cell transmission were effective when used in combination.
- However all of these experiments were done at comparable T cell infection levels between the two transmission modes. In reality cell-cell transmission facilitates high local multiplicity of infection which has been proposed by some as the method by which cell-cell transmission may overcome ART
- However Agosto et al. showed that a viral multiplicity of infection of 25, close to highest detected in HIV cell-cell transmission, did not reduced the efficacy of non-nucleoside analogue reverse transcriptase inhibitors, such as nevirapine and efavirenz suggesting they are multiplicity of infection independent, this feature could be valuable in predicting the effectiveness of drugs against HIV cell-cell transmission.
- It must be noted that significantly fewer drugs were tested for efficacy at MOI 25 than in the cell-cell transmission model, this could be because they didn’t retain efficacy in these conditions. Other studies have shown multiplicity of infection to cause saturation of ART and hence reduced efficacy, seems this what reduces ART efficacy in most cases not the mechanism of spread but the scale it allows.
contribution of CC spread to viral infection
Iwami et al.2015 estimated cell-cell and cell-free spread using a static and shaking culture (in a culture shaken gently cell-cell transmission can be impeded). This was then used to mathematically model HIV infection and it was concluded that cell-cell transmission contributes to ~60% of viral infection and that a complete block of cell-free infection would only have a limited impact on HIV spread.
CC spread and blabs
It has also been suggested to protect HIV particles from humoral immune responses. Li et al 2017. Showed that potency of bNAbs was reduced in cell-cell transmission compared to cell-free transmission. Interestingly it also found that incomplete neutralisation in cell-cell transmission models occurred even with peak bNAb dose.
one virus initiates infection
1 virus initiates infection (Salazar-Gonzalez, 2009)
• A cohort of couples where one partner is known to be infected, but the other uninfected, and they are at high risk of transmission as refuse to use protection -> at some point the uninfected partner is likely to become infected.
• Regular blood samples were taken (regularly – every one or two weeks) and tested for both viraemia and seropositivity. When the uninfected partner becomes infected, retrospective samples are available that allow detection of the very first viral products.
• Ultradeep sequencing of viral genomes was carried out
• Mathematical algorithm then used to derive founder virus (Le et al, 2009)
• Deduced that in large percentage of cases, env sequence in blood of chronically infected partner was found to be identical to that of the transmitted/founder virus identified in the recipient inferred that single virus was responsible for establishing productive infection
1 virus initiates infection rates in different cohorts
80% of heterosexually transmitted infections are initiated by a single virus founder clone (reinforcing the point that it is actually quite hard for virus to infect across the genital mucosa -> often only a single virus is successful, which then initiates the infection). Salazar-Gonzalez et al, 2009 In MSM (men who have sex with men) spread = more likely, since rectal epithelium is more fragile, however, 60% of infections are still initiated by a single virus clone. Li et al, 2010 IVDU (intravenous drug use) transmission = 40% initiated by a single virus clone (much higher risk 🡪 possible to inject multiple virus clones directly into the bloodstream). Bar et al, 2010
leukocyte trafficking in amplification
Study by Deruaz et al.2017 showed blocking of leukocyte trafficking by preventing their response to chemoattractant after vaginal HIV challenge in Bone Liver Thymus (BLT) prevented HIV leaving the genital tract. Suggests transport via leukocytes necessary.
