Allen Ka Loon Cheung

PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice

Viral vector-based vaccines that induce protective CD8+ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non-DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8+ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1-based vaccination potentiated CD8+ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12-producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8+ T cell immunity induced by soluble PD1-based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.

CCR5 antagonist TD-0680 uses a novel mechanism for enhanced potency against HIV-1 entry, cell-mediated infection, and a resistant variant

Background: Maraviroc-resistant HIV-1 posts challenges to CCR5 antagonist discovery. Results: CCR5 antagonist TD-0680 employs a novel mechanism for subnanomolar potency against HIV-1 entry, cell-mediated infection, and a TAK-779/Maraviroc-resistant variant. Conclusion: Distinct binding mode of TD-0680 accounts for its enhanced potency. Significance: Our findings have implications for drug design and developing TD-0680 as an antiretroviral and/or as a microbicide against HIV-1. Regardless of the route of transmission, R5-tropic HIV-1 predominates early in infection, rendering C-C chemokine receptor type 5 (CCR5) antagonists as attractive agents not only for antiretroviral therapy but also for prevention. Here, we report the specificity, potency, and underlying mechanism of action of a novel small molecule CCR5 antagonist, TD-0680. TD-0680 displayed the greatest potency against a diverse group of R5-tropic HIV-1 and SIV strains when compared with its prodrug, TD-0232, the Food and Drug Administration-approved CCR5 antagonist Maraviroc, and TAK-779, with EC50 values in the subnanomolar range (0.09–2.29 nm). Importantly, TD-0680 was equally potent at blocking envelope-mediated cell-cell fusion and cell-mediated viral transmission as well as the replication of a TAK-779/Maraviroc-resistant HIV-1 variant. Interestingly, TD-0232 and TD-0680 functioned differently despite binding to a similar transmembrane pocket of CCR5. Site-directed mutagenesis, drug combination, and antibody blocking assays identified a novel mechanism of action of TD-0680. In addition to binding to the transmembrane pocket, the unique exo configuration of this molecule protrudes and sterically blocks access to the extracellular loop 2 (ECL2) region of CCR5, thereby interrupting the interaction between virus and its co-receptor more effectively. This mechanism of action was supported by the observations of similar TD-0680 potency against CD4-dependent and -independent SIV strains and by molecular docking analysis using a CCR5 model. TD-0680, therefore, merits development as an anti-HIV-1 agent for therapeutic purposes and/or as a topical microbicide for the prevention of sexual transmission of R5-tropic HIV-1.

Vaccine-elicited CD8+ T cells cure mesothelioma by overcoming tumor-induced immunosuppressive environment

Eradicating malignant tumors by vaccine-elicited host immunity remains a major medical challenge. To date, correlates of immune protection remain unknown for malignant mesothelioma. In this study, we demonstrated that antigen-specific CD8(+) T-cell immune response correlates with the elimination of malignant mesothelioma by a model PD-1-based DNA vaccine. Unlike the nonprotective tumor antigen WT1-based DNA vaccines, the model vaccine showed complete and long-lasting protection against lethal mesothelioma challenge in immunocompetent BALB/c mice. Furthermore, it remained highly immunogenic in tumor-bearing animals and led to therapeutic cure of preexisting mesothelioma. T-cell depletion and adoptive transfer experiments revealed that vaccine-elicited CD8(+) T cells conferred to the protective efficacy in a dose-dependent way. Also, these CD8(+) T cells functioned by releasing inflammatory IFNγ and TNFα in the vicinity of target cells as well as by initiating TRAIL-directed tumor cell apoptosis. Importantly, repeated DNA vaccinations, a major advantage over live-vectored vaccines with issues of preexisting immunity, achieve an active functional state, not only preventing the rise of exhausted PD-1(+) and Tim-3(+) CD8(+) T cells but also suppressing tumor-induced myeloid-derived suppressive cells and Treg cells, with the frequency of antigen-specific CD8(+) T cells inversely correlating with tumor mass. Our results provide new insights into quantitative and qualitative requirements of vaccine-elicited functional CD8(+) T cells in cancer prevention and immunotherapy.

Brain Invasion by CD4+ T Cells Infected with a Transmitted/Founder HIV-1BJZS7 During Acute Stage in Humanized Mice

Human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) is one of the common causes of cognitive dysfunction and morbidity among infected patients. However, to date, it remains unknown if a transmitted/founder (T/F) HIV-1 leads to neurological disorders during acute phase of infection. Since it is impossible to answer this question in humans, we studied NOD.Cg-Prkdc scid Il2rgtm1Wjl/SzJ mice (NSG) reconstituted with human PBMC (NSG-HuPBL), followed by the peritoneal challenge with the chronic HIV-1JR-FL and the T/F HIV-1BJZS7, respectively. By measuring viral load, P24 antigenemia and P24+ cells in peripheral blood and various tissue compartments, we found that systemic infections were rapidly established in NSG-HuPBL mice by both HIV-1 strains. Although comparable peripheral viral loads were detected during acute infection, the T/F virus appeared to cause less CD4+ T cell loss and less numbers of infected cells in different organs and tissue compartments. Both viruses, however, invaded brains with P24+/CD3+ T cells detected primarily in meninges, cerebral cortex and perivascular areas. Critically, brain infections with HIV-1JR-FL but not with HIV-1BJZS7 resulted in damaged neurons together with activated microgliosis and astrocytosis as determined by significantly increased numbers of Iba1+ microglial cells and GFAP+ astrocytes, respectively. The increased Iba1+ microglia was correlated positively with levels of P24 antigenemia and negatively with numbers of NeuN+ neurons in brains of infected animals. Our findings, therefore, indicate the establishment of two useful NSG-HuPBL models, which may facilitate future investigation of mechanisms underlying HIV-1-induced microgliosis and astrocytosis.

Gut-homing Δ42PD1+Vδ2 T cells promote innate mucosal damage via TLR4 during acute HIV type 1 infection

The innate immune cells underlying mucosal inflammatory responses and damage during acute HIV-1 infection remain incompletely understood. Here, we report a Vδ2 subset of gut-homing γδ T cells with significantly upregulated Δ42PD1 (a PD1 isoform) in acute (~20%) HIV-1 patients compared to chronic HIV-1 patients (~11%) and healthy controls (~2%). The frequency of Δ42PD1+Vδ2 cells correlates positively with plasma levels of pro-inflammatory cytokines and fatty-acid-binding protein before detectable lipopolysaccharide in acute patients. The expression of Δ42PD1 can be induced by in vitro HIV-1 infection and is accompanied by high co-expression of gut-homing receptors CCR9/CD103. To investigate the role of Δ42PD1+Vδ2 cells in vivo, they were adoptively transferred into autologous humanized mice, resulting in small intestinal inflammatory damage, probably due to the interaction of Δ42PD1 with its cognate receptor Toll-like receptor 4 (TLR4). In addition, blockade of Δ42PD1 or TLR4 successfully reduced the cytokine effect induced by Δ42PD1+Vδ2 cells in vitro, as well as the mucosal pathological effect in humanized mice. Our findings have therefore uncovered a Δ42PD1–TLR4 pathway exhibited by virus-induced gut-homing Vδ2 cells that may contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Δ42PD1+Vδ2 cells may serve as a target for the investigation of diseases with mucosal inflammation.During acute HIV type 1 infection, a subset of γδ T cells that express Δ42PD1 are shown to home to the gut, where they activate innate immunity and inflammation through direct interaction of Δ42PD1 with Toll-like receptor 4. Blockade of this pathway reduces mucosal damage.

Latent human cytomegalovirus enhances HIV-1 infection in CD34+ progenitor cells

Individuals who have been preinfected by human cytomegalovirus (HCMV) are more prone to AIDS disease progression after subsequent HIV-1 infection but the underlying mechanism remains elusive. HCMV is a ubiquitous DNA virus that commonly establishes lifelong latent infection in CD34+ progenitor cells, where latency-specific HCMV genes may modulate host restriction to HIV-1 infection. To test this hypothesis, we studied progenitor cells that are known to resist replicative HIV-1 infection because of the intrinsic expression of host restriction factors. Interestingly, in primary CD34+ cells undergoing latent HCMV infection, an enhanced level of HIV-1 proviral DNA and replication was observed as measured by digital polymerase chain reaction, quantitative polymerase chain reaction, and Gag expression, and confirmed using dual-reporter pseudovirus encoding X4- or R5-tropic envelope and T-cell transfer. This phenomenon may be partially explained by the upregulation of HIV-1 entry coreceptors, including chemokine receptors CXCR4 and CCR5, but not of the primary receptor CD4. Furthermore, latent HCMV infection downregulated the expression of HIV-1 restriction factors SAMHD1, APOBEC3G, tetherin, and Mx2 in CD34+ progenitor cells, which may confer to enhanced HIV-1 infection. However, this enhancement was abrogated when ultraviolet-inactivated HCMV was used for comparison, suggesting that expression of latent HCMV genes is essential for this effect. Importantly, HCMV gB and HIV-1 p24 can be detected in the same cell by immunofluorescence and flow cytometry; therefore, the establishment of HCMV latency in CD34+ cells likely leads to host cell gene modulation that favors HIV-1 infection.

Monoclonal antibodies specific to human Δ42PD1: A novel immunoregulator potentially involved in HIV-1 and tumor pathogenesis.

We recently reported the identification of Δ42PD1, a novel alternatively spliced isoform of human PD1 that induces the production of pro-inflammatory cytokines from human peripheral blood mononuclear cells and enhances HIV-specific CD8+ T cell immunity in mice when engineered in a fusion DNA vaccine. The detailed functional study of Δ42PD1, however, has been hampered due to the lack of a specific monoclonal antibody (mAb). In this study, we generated 2 high-affinity mAbs, clones CH34 (IgG2b) and CH101 (IgG1), from Δ42PD1-immunized mice. They recognize distinct domains of Δ42PD1 as determined by a yeast surface-displaying assay and ELISA. Moreover, they recognize native Δ42PD1 specifically, but not PD1, on cell surfaces by both flow cytometry and immunohistochemical assays. Δ42PD1 appeared to be expressed constitutively on healthy human CD14+ monocytes, but its level of expression was down-regulated significantly during chronic HIV-1 infection. Since the level of Δ42PD1 expression on CD14+ monocytes was negatively correlated with the CD4 count of untreated patients in a cross-sectional study, Δ42PD1 may play a role in HIV-1 pathogenesis. Lastly, when examining Δ42PD1 expression in human esophageal squamous-cell carcinoma tissues, we found high-level expression of Δ42PD1 on a subset of tumor-infiltrating T cells. Our study, therefore, resulted in 2 Δ42PD1-specific mAbs that can be used to further investigate Δ42PD1, a novel immune regulatory protein implicated in HIV-1 and tumor pathogenesis as well as other immune diseases.

DNA prime/MVTT boost regimen with HIV-1 mosaic Gag enhances the potency of antigen-specific immune responses

HIV-1 diversity and latent reservoir are the major challenges for the development of an effective AIDS vaccine. It is well indicated that Gag-specific CD8+ T cells serve as the dominant host immune surveillance for HIV-1 control, but it still remains a challenge for vaccine design to induce broader and stronger cytotoxic T cell immunity against the virus. Genetic variation of the HIV-1 gag gene across different clades is one of the reasons for the reduction of antigenic epitope coverage. Here, we report an immunization strategy with heterologous vaccines expressing a mosaic Gag antigen aimed to increase antigenic breadth against a wider spectrum of HIV-1 strains. Priming using a DNA vaccine via in vivo electroporation, followed by boosting with a live replication-competent modified vaccinia TianTan (MVTT) vectored vaccine, elicited greater and broader protective Gag-specific immune responses in mice. Compared to DNA or MVTT homologous immunization, the heterologous DNA/MVTT vaccination resulted in higher frequencies of broadly reactive, Gag-specific, polyfunctional, long-lived cytotoxic CD8+ T cells, as well as increased anti-Gag antibody titer. Importantly, the DNA/MVTT heterologous vaccination induced protection against EcoHIV and mesothelioma AB1-Gag challenges. In summary, the stronger protective Gag-specific immunity induced by the heterologous regimen using two safe vectors shows promise for further development to enhance anti-HIV-1 immunity. Our study has important implications for immunogen design and the development of an effective HIV-1 heterologous vaccination strategy.

HIV-1 genetic transmission networks among men who have sex with men in Kunming, China

Background Yunnan has the greatest share of reported human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) cases in China. In recent years, HIV prevalence and incidence remained stubbornly high in men who have sex with men (MSM). To follow the dynamics of the HIV-1 epidemic among MSM, HIV-1 genetic characteristics and genetic transmission networks were investigated. Methods Blood samples from 190 newly diagnosed HIV-1 cases among MSM were continuously collected at fixed sites from January 2013 to December 2015 in Kunming City, Yunnan Province. Partial gag, pol and env genes were sequenced and used for phylogenetic and genotypic drug resistance analyses. The genetic characteristics of the predominant HIV-1 strains were analyzed by the Bayesian Markov Chain Monte Carlo (MCMC) method. The genetic transmission networks were identified with a genetic distance of 0.03 substitutions/site and 90% bootstrap support. Results Among the 190 HIV-1 positive MSM reported during 2013–2105, various genotypes were identified, including CRF01_AE (45.3%), CRF07_BC (35.8%), unique recombinant forms (URFs) (11.6%), CRF08_BC (3.2%), CRF55_01B (2.1%), subtype B (1.6%) and CRF59_01B (0.5%). The effective population sizes (EPS) for CRF01_AE and CRF07_BC increased exponentially from approximately 2001–2010 and 2005–2009, respectively. Genetic transmission networks were constructed with 308 pol sequences from MSM diagnosed during 2010–2015. Of the 308 MSM, 109 (35.4%) were identified in 38 distinct clusters. Having multiple male partners was associated with a high probability of identification in the genetic transmission networks. Of the 38 clusters, 27 (71.1%) contained individuals diagnosed in different years. Of the 109 individuals in the networks, 26 (23.9%) had ≥2 potential transmission partners (≥2 links). The proportion of MSM with ≥2 links was higher among those diagnosed from 2010–2012. The constituent ratios of their potential transmission partners by areas showed no significant difference among MSM from Kunming, other cities in Yunnan and other provinces. Additionally, surveillance drug resistance mutations (SDRMs) were identified in 5% of individuals. Conclusion This study revealed the various HIV-a genotypes circulating among MSM in Kunming. MSM with more partners were more easily detected in transmission networks, and early-diagnosed MSM remained active in transmission networks. These findings suggested that the routine interventions should be combined with HIV testing and linkage to care and early antiretroviral therapy among HIV-positive MSM.