Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors.

BACKGROUND: Chimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs. METHODS: We used Humabody VH domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) VH and mesothelin (MSLN) VH sequences and redirect T cell with VH based-CAR. The antitumor activity and mode of action of PSMA VH and MSLN VH were evaluated in vitro and in vivo compared with the traditional scFv-based CARs. RESULTS: Human VH domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. VH modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression. CONCLUSIONS: Fully human antibody VH domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, VH domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.

Inhibition of post-surgery tumour recurrence via a hydrogel releasing CAR-T cells and anti-PDL1-conjugated platelets.

The immunosuppressive microenvironment of solid tumours reduces the antitumour activity of chimeric antigen receptor T cells (CAR-T cells). Here, we show that the release-through the implantation of a hyaluronic acid hydrogel-of CAR-T cells targeting the human chondroitin sulfate proteoglycan 4, polymer nanoparticles encapsulating the cytokine interleukin-15 and platelets conjugated with the checkpoint inhibitor programmed death-ligand 1 into the tumour cavity of mice with a resected subcutaneous melanoma tumour inhibits the local recurrence of the tumour as well as the growth of distant tumours, through the abscopal effect. The hydrogel, which functions as a reservoir, facilitates the enhanced distribution of the CAR-T cells within the surgical bed, and the inflammatory microenvironment triggers platelet activation and the subsequent release of platelet-derived microparticles. The post-surgery local delivery of combination immunotherapy through a biocompatible hydrogel reservoir could represent a translational route for preventing the recurrence of cancers with resectable tumours.

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

HIV-1 Central Nervous System Compartmentalization and Cytokine Interplay in Non-Subtype B HIV-1 Infections in Nigeria and Malawi.

HIV-1 compartmentalization in the central nervous system (CNS) and its contribution to neurological disease have been well documented. Previous studies were conducted among people infected with subtypes B or C where CNS compartmentalization has been observed when comparing viral sequences in the blood to virus in cerebrospinal fluid (CSF). However, little is known about CNS compartmentalization in other HIV-1 subtypes. Using a deep sequencing approach with Primer ID, we conducted a cross-sectional study among Nigerian and Malawian HIV-1 cohorts with or without fungal Cryptococcus infection diagnosed as cryptococcal meningitis (CM) to determine the extent of CSF/CNS compartmentalization with CM. Paired plasma and CSF samples from 45 participants were also analyzed for cytokine/chemokine levels. Viral populations comparing virus in the blood and the CSF ranged from compartmentalized to equilibrated, including minor or partial compartmentalization or clonal amplification of a single viral sequence. The frequency of compartmentalized viral populations in the blood and CSF was similar between the CM- and CM+ participants. We confirmed the potential to see compartmentalization with subtype C infection and have also documented CNS compartmentalization of an HIV-1 subtype G infection. Cytokine profiles indicated a proinflammatory environment, especially within the CSF/CNS. However, sCD163 was suppressed in the CSF in the presence of CM, perhaps due to elevated levels of IL-4, which were also a feature of the cytokine profile, showing a distinct cytokine profile with CM.

Rationally designed carbohydrate-occluded epitopes elicit HIV-1 Env-specific antibodies.

An array of carbohydrates masks the HIV-1 surface protein Env, contributing to the evasion of humoral immunity. In most HIV-1 isolates 'glycan holes' occur due to natural sequence variation, potentially revealing the underlying protein surface to the immune system. Here we computationally design epitopes that mimic such surface features (carbohydrate-occluded neutralization epitopes or CONE) of Env through 'epitope transplantation', in which the target region is presented on a carrier protein scaffold with preserved structural properties. Scaffolds displaying the four CONEs are examined for structure and immunogenicity. Crystal structures of two designed proteins reflect the computational models and accurately mimic the native conformations of CONEs. The sera from rabbits immunized with several CONE immunogens display Env binding activity. Our method determines essential structural elements for targets of protective antibodies. The ability to design immunogens with high mimicry to viral proteins also makes possible the exploration of new templates for vaccine development.

Photothermal Therapy Promotes Tumor Infiltration and Antitumor Activity of CAR T Cells.

Chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR T cells) show modest therapeutic efficacy in solid tumors. The desmoplastic structure of the tumor and the immunosuppressive tumor microenvironment usually account for the reduced efficacy of CAR T cells in solid tumors. Mild hyperthermia of the tumor reduces its compact structure and interstitial fluid pressure, increases blood perfusion, releases antigens, and promotes the recruitment of endogenous immune cells. Therefore, the combination of mild hyperthermia with the adoptive transfer of CAR T cells can potentially increase the therapeutic index of these cells in solid tumors. It is found that the chondroitin sulfate proteoglycan-4 (CSPG4)-specific CAR T cells infused in Nod scid gamma mice engrafted with the human melanoma WM115 cell line have superior antitumor activity after photothermal ablation of the tumor. The findings suggest that photothermal therapy facilitates the accumulation and effector function of CAR T cells within solid tumors.

Characterization of the Transmitted Virus in an Ongoing HIV-1 Epidemic Driven by Injecting Drug Use.

Understanding features of the HIV-1 transmission process has the potential to inform biological interventions for prevention. We have examined the transmitted virus in a cohort of people who inject drugs and who are at risk of HIV-1 infection through blood contamination when injecting in a group. This study focused on seven newly infected participants in St. Petersburg, Russia, who were in acute or early infection. We used end-point dilution polymerase chain reaction to amplify single viral genomes to assess the complexity of the transmitted virus. We also used deep sequencing to further assess the complexity of the virus. We interpret the results as indicating that a single viral variant was transmitted in each case, consistent with a model where the exposure to virus during transmission was limited. We also looked at phenotypic properties of the viral Env protein in isolates from acute and chronic infection. Although differences were noted, there was no consistent pattern that distinguished the transmitted variants. Similarly, despite the reduced genetic heterogeneity of the more recent subtype A HIV-1 epidemic in St. Petersburg, we did not see reduced variance in the neutralization properties compared to isolates from the more mature subtype C HIV-1 epidemic. Finally, in looking at members of injecting groups related to the acute HIV-1 infection/early subjects, we found examples of sequence linkage consistent with ongoing and rapid spread of HIV-1 in these groups. These studies emphasize the dynamic nature of this epidemic and reinforce the idea that improved prevention methods are needed.

A Cohort Approach to Real-Time Detection of Acute HIV Infections Among People Who Inject Drugs in St. Petersburg, Russia.

To detect acute HIV infections (AHIs) in real time among people who inject drugs (PWID) in St. Petersburg, Russia and to test the feasibility of this approach. Prospective cohort study. One hundred seronegative or acutely HIV-infected at screening PWID were enrolled and followed until the end of the 12-month pilot period. Each participant was evaluated, tested, and counseled for HIV monthly. Two HIV tests were used: HIV antibody and HIV RNA PCR. If diagnosed with AHI, participants were followed weekly for a month; then, monthly for 3 months; and then, quarterly for the duration of the follow-up period. HIV risk behavior was assessed at each study visit. Most enrolled PWID were 30-39 years old, male, completed high school or more, not employed full-time, heroin users, and frequently shared injection paraphernalia. AHI prevalence at screening was 1.8% [95% confidence interval (CI): 0.4, 5.5]. Three participants with AHI at enrollment represented 3% (95% CI: 0.6, 8.5) of the 100 participants who consented to enroll. Among the HIV-uninfected participants (n = 97), the AHI incidence over time was 9.3 per 100 person-years. Persons with AHI were more likely to report alcohol intoxication within the prior 30 days. This was the first study to detect AHI using a cohort approach. The approach proved to be feasible: recruitment, retention, AHI detection, and virological endpoints were successfully reached. A cost analysis in a real-world setting would be required to determine if this strategy could be brought to scale. The study revealed continued high HIV incidence rate among PWID in St. Petersburg, Russia and the importance of prevention and treatment programs for this group.

Treatment as Prevention: Characterization of Partner Infections in the HIV Prevention Trials Network 052 Trial.

HIV Prevention Trials Network 052 demonstrated that antiretroviral therapy (ART) prevents HIV transmission in serodiscordant couples. HIV from index-partner pairs was analyzed to determine the genetic linkage status of partner infections. Forty-six infections were classified as linked, indicating that the index was the likely source of the partner's infection. Lack of viral suppression and higher index viral load were associated with linked infection. Eight linked infections were diagnosed after the index started ART: 4 near the time of ART initiation and 4 after ART failure. Linked infections were not observed when the index participant was stably suppressed on ART.

Phenotypic Correlates of HIV-1 Macrophage Tropism.

UNLABELLED: HIV-1 is typically CCR5 using (R5) and T cell tropic (T-tropic), targeting memory CD4(+) T cells throughout acute and chronic infections. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize the phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation. IMPORTANCE: HIV-1 typically replicates in CD4(+) T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses evolve and differ from CCR5-using T cell-tropic viruses may provide insights into viral evolution and pathogenesis within the central nervous system. We characterized the HIV-1 env viral entry gene from subject-matched macrophage-tropic and T cell-tropic viruses to identify entry features of macrophage-tropic viruses. We observed several differences between T cell-tropic and macrophage-tropic Env proteins, including functional differences with host CD4 receptor engagement and possible changes in the CD4 binding site and V1/V2 region. We also identified viruses with phenotypes between that of "true" macrophage-tropic and T cell-tropic viruses, which may represent evolutionary intermediates in a multistep process to macrophage tropism.

R5 Macrophage-Tropic HIV-1 in the Male Genital Tract.

The entry tropism of HIV-1 Env proteins from virus isolated from the blood and genital tract of five men with compartmentalized lineages was determined. The Env proteins isolated from the genital tract of subject C018 were macrophage-tropic proteins, while the remaining cloned env genes encoded R5 T cell-tropic proteins. The detection of a macrophage-tropic lineage of HIV-1 within the male genital tract strongly suggests that evolution of macrophage-tropic viruses can occur in anatomically isolated sites outside the central nervous system.

Compartmentalization, Viral Evolution, and Viral Latency of HIV in the CNS.

Human immunodeficiency virus type 1 (HIV-1) infection occurs throughout the body and can have dramatic physical effects, such as neurocognitive impairment in the central nervous system (CNS). Furthermore, examining the virus that resides in the CNS is challenging due to its location and can only be done using samples collected either at autopsy, indirectly form the cerebral spinal fluid (CSF), or through the use of animal models. The unique milieu of the CNS fosters viral compartmentalization as well as evolution of viral sequences, allowing for new cell types, such as macrophages and microglia, to be infected. Treatment must also cross the blood-brain barrier adding additional obstacles in eliminating viral populations in the CNS. These long-lived infected cell types and treatment barriers may affect functional cure strategies in people on highly active antiretroviral therapy (HAART).

Two Independent HIV Epidemics in Saint Petersburg, Russia Revealed by Molecular Epidemiology.

The HIV epidemic in Russia, one of the world's fastest growing, has been concentrated mostly among people who inject drugs (PWID). We sought to explore the epidemiology of the epidemic in St. Petersburg by sampling from the highest risk groups of PWID and men who have sex with men (MSM) and use viral sequencing data to better understand the nature of the city's epidemic. Serological testing confirmed an HIV prevalence among PWID in excess of 40%. All but 1 of 110 PWID whose blood samples were tested for genetic diversity were infected by subtype A virus, specifically by the AFSU strain. The remaining person was infected with a CRF-06cpx recombinant. Analysis of pairwise genetic distance among all PWID studied revealed an average of 3.1% sequence divergence, suggesting clonal introduction of the AFSU strain and/or constraints on sequence divergence. The HIV prevalence was less than 10% among MSM. All 17 sequences from HIV-infected MSM were found to be a clade B virus with a much higher average sequence diversity of 15.7%. These findings suggest two independent epidemics with little overlap between the two highest at-risk populations, which will require different HIV prevention approaches.

A substantial transmission bottleneck among newly and recently HIV-1-infected injection drug users in St Petersburg, Russia.

There are limited data on the genetic complexity of human immunodeficiency virus type 1 (HIV-1) after transmission among a cohort of injection drug users (IDUs). We used single-genome amplification of HIV-1 env to determine the genotypic characteristics of virus among IDUs with acute infection in St Petersburg, Russia. Our results indicate that a single variant was transmitted in a majority of cases (9 of 13 participants), which is analogous to what is observed in sexual transmission. These data are most consistent with a genetic bottleneck during transmission by injection drug use that is due to a small inoculum, which most often results in the transmission of a low-complexity viral population.

Hepatitis C virus infection among drug injectors in St Petersburg, Russia: social and molecular epidemiology of an endemic infection.

AIMS: To understand the epidemiology and transmission patterns of hepatitis C virus (HCV), the predominant blood borne-pathogen infecting injection drug users (IDUs), in a part of the former Soviet Union. DESIGN: Cross-sectional respondent-driven sample of IDUs. SETTING: St Petersburg, Russia. PARTICIPANTS: A total of 387 IDUs were recruited in late 2005 and throughout 2006. MEASUREMENTS: Participants were surveyed to collect demographic, medical and both general and dyad-specific drug injection and sexual behaviors. A blood sample was collected to detect antibodies to hepatitis C and to amplify viral RNA for molecular analysis. The molecular data, including genotypes, were analyzed spatially and linkage patterns were compared to the social linkages obtained by respondent-driven sampling (RDS) for chains of respondents and among the injection dyads. FINDINGS: HCV infection was all but ubiquitous: 94.6% of IDUs were HCV-seropositive. Among the 209 viral sequences amplified, genotype 3a predominated (n = 119, 56.9%), followed by 1b (n = 61, 29.2%) and 1a (n = 25, 11.9%). There was no significant clustering of genotypes spatially. Neither genotypes nor closely related sequences were clustered within RDS chains. Analysis of HCV sequences from dyads failed to find associations of genotype or sequence homology within pairs. CONCLUSIONS: Genotyping reveals that there have been at least five unique introductions of HCV genotypes into the IDU community in St Petersburg. Analysis of prevalent infections does not appear to correlate with the social networks of IDUs, suggesting that simple approaches to link these networks to prevalent infections, rather than incident transmission, will not prove meaningful. On a more positive note, the majority of IDUs are infected with 3a genotype that is associated with sustained virological response to antiviral therapy.

The feasibility of HIV vaccine efficacy trials among Russian injection drug users.

IDU exposure remains a primary driver of the Russian HIV epidemic, and recent incidence data provide little evidence that this epidemic is slowing. While there are multiple important challenges that need to be further explored before starting vaccine trials, most importantly access to evidence-based drug treatment services for trial participants, the current context of high HIV incidence and low genetic diversity of HIV strains, suggests the need for intensified prevention strategies and supports the feasibility of mounting efficacy trials of HIV vaccines among IDUs in the Russian Federation.

Immunogenicity of candidate DNA vaccine based on subtype A of human immunodeficiency virus type 1 predominant in Russia.

Human immunodeficiency virus (HIV)-1 subtype A strains circulating among the majority of HIVinfected individuals in the former Soviet Union (FSU) countries demonstrate low genetic diversity. The consensus sequence of the FSU region-specific isolate has been used for the candidate DNA vaccine development. We constructed recombinant plasmids with four viral genes: env (gp140), gag, pol (reverse transcriptase), and nef. We immunized BALB/c mice intramuscularly using equimolar mixture of four recombinant plasmids, and observed significant cytotoxic T lymphocyte response and specific CD8(+) cell production against cells presenting HIV-1 peptides. Overall, the Th1 pathway of immune response clearly dominated. Immunological properties of this candidate DNA vaccine against HIV-1 suggest the possibility of its further study in clinical trials.