Janelle R. Salkowitz

Highly potent, fully recombinant anti-HIV chemokines: Reengineering a low-cost microbicide

New prevention strategies for use in developing countries are urgently needed to curb the worldwide HIV/AIDS epidemic. The N-terminally modified chemokine PSC-RANTES is a highly potent entry inhibitor against R5-tropic HIV-1 strains, with an inhibitory mechanism involving long-term intracellular sequestration of the HIV coreceptor, CCR5. PSC-RANTES is fully protective when applied topically in a macaque model of vaginal HIV transmission, but it has 2 potential disadvantages related to further development: the requirement for chemical synthesis adds to production costs, and its strong CCR5 agonist activity might induce local inflammation. It would thus be preferable to find a recombinant analogue that retained the high potency of PSC-RANTES but lacked its agonist activity. Using a strategy based on phage display, we set out to discover PSC-RANTES analogs that contain only natural amino acids. We sought molecules that retain the potency and inhibitory mechanism of PSC-RANTES, while trying to reduce CCR5 signaling to as low a level as possible. We identified 3 analogues, all of which exhibit in vitro potency against HIV-1 comparable to that of PSC-RANTES. The first, 6P4-RANTES, resembles PSC-RANTES in that it is a strong agonist that induces prolonged intracellular sequestration of CCR5. The second, 5P12-RANTES, has no detectable G protein-linked signaling activity and does not bring about receptor sequestration. The third, 5P14-RANTES, induces significant levels of CCR5 internalization without detectable G protein-linked signaling activity. These 3 molecules represent promising candidates for further development as topical HIV prevention strategies.

CCR5 promoter polymorphism determines macrophage CCR5 density and magnitude of HIV-1 propagation in vitro.

The common CCR5 promoter polymorphism at position -2459 (A/G) has been associated with differences in the rate of progression to AIDS, where HIV-1-infected individuals with the CCR5 -2459 G/G genotype exhibit slower disease progression than those with the A/A genotype. Mechanisms underlying the relationship between these polymorphisms and disease progression are not known. Here through in vitro infection of peripheral blood mononuclear cells obtained from healthy Caucasian blood donors with macrophage-tropic HIV-1 isolates we observed low, medium, and high viral propagation in association with G/G, A/G, and A/A promoter genotypes, respectively. Flow cytometric analysis of unstimulated CD14+ monocytes from these same donors revealed a similar hierarchy of CCR5 receptor density in association with promoter genotypes. Finally, PBMC from persons with the G/G promoter polymorphism produced higher levels of beta-chemokines after in vitro stimulation. Thus, the CCR5 -2459 (A/G) promoter polymorphism determines CCR5 expression and predicts the magnitude of HIV-1 propagation in vitro. These findings may provide important insight regarding the regulation of mechanisms that influence the rate of HIV-1 propagation and progression to AIDS.

Immune responses to hepatitis C and non-hepatitis C antigens in hepatitis C virus infected and HIV-1 coinfected patients

ObjectiveTo characterize immune phenotype and function in hepatitis C virus (HCV) infection in the presence and absence of HIV-1 infection. DesignCross-sectional comparison among controls (group A), patients with HCV infection (group B), HCV–HIV-1 coinfected patients (group C), coinfected patients receiving treatment for HIV-1 (group D), and untreated HIV-1 infected patients (group E). MethodsFlow cytometric analysis for lymphocyte phenotypes, lymphocyte proliferation and cytokine production by ELISPOT. ResultsHCV infected patients tended to have an increased percentage of activated (CD38, HLA-DR) CD8 cells (group A, 2 ± 1.4%; group B, 6 ± 3.9%;P = 0.08). Proliferative responses to non-HCV antigens were comparable in group A and group B subjects. A greater proportion of group B patients had stimulation indices (SI) > 3 to the HCV protein NS3 compared to group C and D patients (67%, 0%, and 11% respectively;P < 0.003), but only two patients in group B had SI ⩾ 5. The SI to NS3 was significantly higher in group B patients [median, 4; interquartile range (IQR), 3–9) than in group C (median, 2; IQR, 1–3;P < 0.04) or group D (median, 1; IQR, 1–4;P < 0.009) patients. Plasma HCV RNA levels correlated directly with alanine aminotransferase levels (ρ, 0.52;P < 0.05) and inversely with the number of CD4 lymphocytes (ρ, −0.55;P < 0.009) and proliferation to NS3 (ρ, −0.55;P < 0.009). ConclusionsLymphocytes of HCV infected patients show weak proliferative responses to HCV antigens while responses to other antigens are preserved. Infection with HIV-1 potentiates this deficiency. Poor CD4 T cell responses to HCV are associated with and may determine the failure to control HCV propagation.

Evolution of CCR5 Use before and during Coreceptor Switching

ABSTRACT The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.

Impaired Monocyte Maturation in Response to CpG Oligodeoxynucleotide Is Related to Viral RNA Levels in Human Immunodeficiency Virus Disease and Is at Least Partially Mediated by Deficiencies in Alpha/Beta Interferon Responsiveness and Production

ABSTRACT The biological activity of CpG oligodeoxynucleotide 2216 (ODN2216), a Toll-like receptor 9 agonist, was investigated with monocytes from human immunodeficiency virus (HIV)-negative and HIV-positive (HIV+) donors. Exposure of peripheral blood mononuclear cells to CpG ODN2216 led to decreased expression of the monocyte marker CD14 and increased expression of the dendritic cell marker CD83, as well as increased expression of HLA-DR, CD40, CD80, and CD86 among the monocytes. Several features of the CpG ODN-induced maturation were diminished in monocytes from HIV+ donors, and these deficiencies were related to increased viremia but not to CD4 cell counts. Alpha interferon (IFN-α) was implicated as at least a partial mediator of the CpG ODN-induced monocyte maturation. Reduced production of IFN-α in response to CpG ODN and reduced frequencies of plasmacytoid dendritic cells, the principal IFN-α-producing cell type in peripheral blood, were observed in peripheral blood mononuclear cells from HIV+ donors. These deficiencies also were related to levels of plasma HIV RNA. Responses of monocytes from HIV+ donors to direct stimulation with IFN-α also were partially impaired. Thus, reduced production of IFN-α and reduced IFN-α responsiveness may contribute to diminished functional responses to CpG ODN in HIV disease. Application of CpG ODNs in HIV disease for adjuvant or immunoregulatory purposes may be particularly useful for HIV+ donors without high-level viremia.

Enhanced induction of HIV-specific cytotoxic T lymphocytes by dendritic cell-targeted delivery of SOCS-1 siRNA.

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8(+) T cells from healthy donors. Finally, stimulation of CD8(+) T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8+ T cells from healthy donors. Finally, stimulation of CD8+ T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.

Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

HLA‐A*0201‐restricted virus‐specific CD8+ CTL do not appear to control HIV effectively in vivo. To enhance the immunogenicity of a highly conserved subdominant epitope, TV9 (TLNAWVKVV, p24 Gag19–27), mimotopes were designed by screening a large combinatorial nonapeptide library with TV9‐specific CTL primed in vitro from healthy donors. A mimic peptide with a low binding affinity to HLA‐A*0201, TV9p6 (KINAWIKVV), was studied further. Parallel cultures of in vitro‐primed CTL showed that TV9p6 consistently activated cross‐reactive and equally functional CTL as measured by cytotoxicity, cytokine production and suppression of HIV replication in vitro. Comparison of TCRB gene usage between CTL primed from the same donors with TV9 or TV9p6 revealed a degree of clonal overlap in some cases and an example of a conserved TCRB sequence encoded distinctly at the nucleotide level between individuals (a “public” TCR); however, in the main, distinct clonotypes were recruited by each peptide antigen. These findings indicate that mimotopes can mobilize functional cross‐reactive clonotypes that are less readily recruited from the naïve T‐cell pool by the corresponding WT epitope. Mimotope‐induced repertoire diversification could potentially override subdominance under certain circumstances and enhance vaccine‐induced responses to conserved but poorly immunogenic determinants within the HIV proteome.

Differences in the expression of histocompatibility antigen-dr and in anti-mycobacterial activity of monocytes from Hiv-infected individuals

This study assesses the changes in the expression of histocompatibility antigen (HLA)-DR by mononuclear phagocytes from HIV-infected individuals. Overnight culture of monocytes resulted in an increase in HLA-DR expression by monocytes from uninfected individuals. In contrast, the expression of HLA-DR by monocytes from HIV-infected patients decreased spontaneously and was most pronounced in patients with clinical AIDS. We also found that Mycobacterium avium grew within monocytes from patients infected with HIV. The correlation between major histocompatibility complex (MHC) class II expression and Mycobacterial growth which has been reported in mice was not observed in monocytes from HIV-infected patients.

Animal models for AIDS pathogenesis

Publisher Summary Individuals who harbor naturally attenuated HIV do not escape disease, but, like the rhesus macaques, they experience a delay in its onset. In order for a live attenuated vaccine to succeed, additional control or elimination of persistent proviral copies is necessary. The primate lentiviruses have differential pathogenesis; not all combinations of virus and hosts produce disease. Pathogenesis is a function of both viral and host determinants. Even within a species susceptible to disease caused by an AIDS virus, variability in manifestations of the disease exists. A molecular cloned virus contains the total genetic information for the virus on a proviral DNA copy. That copy can, by itself, program the production of the virus; in fundamental nature the virus life cycle can be started in the absence of complete virus. Thus, pure DNA encoding viral information can be used to test whether the virus causes AIDS or whether some contaminating substance copurifying in the virus preparation is necessary to produce the disease.

P17-24. Interfering overlapping epitopes contribute to the subdominance of an HLA-A2-restricted HIV Gag-specific epitope

Methods To detect novel subdominant determinants in HIV-1 Gag, we primed CD8+ T cells from eight seronegative donors with autologous dendritic cells transduced to express Gag. T cells were re-stimulated weekly with monocytes pulsed with 123 15-mer overlapping peptides (OLPs) spanning Gag. Responses were identified with OLP matrix pools followed by interrogation at the single OLP level in responsive pools using IFN-γ ELISPOT assays.