Checkpoint inhibitor-expressing lentiviral vaccine suppresses tumor growth in preclinical cancer models.

BACKGROUND: While immunotherapy has been highly successful for the treatment of some cancers, for others, the immune response to tumor antigens is weak leading to treatment failure. The resistance of tumors to checkpoint inhibitor therapy may be caused by T cell exhaustion resulting from checkpoint activation. METHODS: In this study, lentiviral vectors that expressed T cell epitopes of an experimentally introduced tumor antigen, ovalbumin, or the endogenous tumor antigen, Trp1 were developed. The vectors coexpressed CD40 ligand (CD40L), which served to mature the dendritic cells (DCs), and a soluble programmed cell death protein 1 (PD-1) microbody to prevent checkpoint activation. Vaccination of mice bearing B16.OVA melanomas with vector-transduced DCs induced the proliferation and activation of functional, antigen-specific, cytolytic CD8 T cells. RESULTS: Vaccination induced the expansion of CD8 T cells that infiltrated the tumors to suppress tumor growth. Vector-encoded CD40L and PD-1 microbody increased the extent of tumor growth suppression. Adoptive transfer demonstrated that the effect was mediated by CD8 T cells. Direct injection of the vector, without the need for ex vivo transduction of DCs, was also effective. CONCLUSIONS: This study suggests that therapeutic vaccination that induces tumor antigen-specific CD8 T cells coupled with a vector-expressed checkpoint inhibitor can be an effective means to suppress the growth of tumors that are resistant to conventional immunotherapy.

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells.

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

Vectored immunoprophylaxis and treatment of SARS-CoV-2 infection in a preclinical model.

Vectored immunoprophylaxis was first developed as a means of establishing engineered immunity to HIV using an adenoassociated viral vector expressing a broadly neutralizing antibody. We applied this concept to establish long-term prophylaxis against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mouse model using adenoassociated virus and lentiviral vectors expressing a high-affinity angiotensin-converting enzyme 2 (ACE2) decoy. Administration of decoy-expressing (adenoassociated virus) AAV2.retro and AAV6.2 vectors by intranasal instillation or intramuscular injection protected mice against high-titered SARS-CoV-2 infection. AAV and lentiviral vectored immunoprophylaxis was durable and was active against SARS-CoV-2 Omicron subvariants. The AAV vectors were also effective therapeutically when administered postinfection. Vectored immunoprophylaxis could be of value for immunocompromised individuals for whom vaccination is not practical and as a means to rapidly establish protection from infection. Unlike monoclonal antibody therapy, the approach is expected to remain active despite continued evolution viral variants.

Vectored Immunoprophylaxis and Treatment of SARS-CoV-2 Infection.

Vectored immunoprophylaxis was first developed as a means to establish engineered immunity to HIV through the use of an adeno-associated viral vector expressing a broadly neutralizing antibody. We have applied this concept to establish long-term prophylaxis against SARS-CoV-2 by adeno-associated and lentiviral vectors expressing a high affinity ACE2 decoy receptor. Administration of decoy-expressing AAV vectors based on AAV2.retro and AAV6.2 by intranasal instillation or intramuscular injection protected mice against high-titered SARS-CoV-2 infection. AAV and lentiviral vectored immunoprophylaxis was durable and active against recent SARS-CoV-2 Omicron subvariants. The AAV vectors were also effective when administered up to 24 hours post-infection. Vectored immunoprophylaxis could be of value for immunocompromised individuals for whom vaccination is not practical and as a means to rapidly establish protection from infection. Unlike monoclonal antibody therapy, the approach is expected to remain active despite continued evolution viral variants.

Directly injected lentiviral vector-based T cell vaccine protects mice against acute and chronic viral infection.

Lentiviral vector-based dendritic cell vaccines induce protective T cell responses against viral infection and cancer in animal models. In this study, we tested whether preventative and therapeutic vaccination could be achieved by direct injection of antigen-expressing lentiviral vector, obviating the need for ex vivo transduction of dendritic cells. Injected lentiviral vector preferentially transduced splenic dendritic cells and resulted in long-term expression. Injection of a lentiviral vector encoding an MHC class I-restricted T cell epitope of lymphocytic choriomeningitis virus (LCMV) and CD40 ligand induced an antigen-specific cytolytic CD8+ T lymphocyte response that protected the mice from infection. The injection of chronically infected mice with a lentiviral vector encoding LCMV MHC class I and II T cell epitopes and a soluble programmed cell death 1 microbody rapidly cleared the virus. Vaccination by direct injection of lentiviral vector was more effective in sterile alpha motif and HD-domain containing protein 1-knockout (SAMHD1-knockout) mice, suggesting that lentiviral vectors containing Vpx, a lentiviral protein that increases the efficiency of dendritic cell transduction by inducing the degradation of SAMHD1, would be an effective strategy for the treatment of chronic disease in humans.

Neutralization of SARS-CoV-2 Variants by mRNA and Adenoviral Vector Vaccine-Elicited Antibodies.

The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine effectiveness. Here, neutralizing antibody titers elicited by mRNA-based and adenoviral vector-based vaccines against variant pseudotyped viruses were measured. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals had less neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest a potential benefit by second immunization following Ad26.COV2.S to increase protection from current and future variants.

High-titer neutralization of Mu and C.1.2 SARS-CoV-2 variants by vaccine-elicited antibodies of previously infected individuals.

Recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants Mu and C.1.2 have spike proteins with mutations that may confer resistance to natural and vaccine-elicited antibodies. Analysis of neutralizing antibody titers in the sera of vaccinated individuals without previous history of infection and from convalescent individuals show partial resistance of the viruses. In contrast, sera from individuals with a previous history of SARS-CoV-2 infection who were subsequently vaccinated neutralize variants with titers 4- to 11-fold higher, providing a rationale for vaccination of individuals with previous infection. The heavily mutated C.1.2 spike is the most antibody neutralization-resistant spike to date; however, the avidity of C.1.2 spike protein for angiotensin-converting enzyme 2 (ACE2) is low. This finding suggests that the virus evolved to escape the humoral response but has a decrease in fitness, suggesting that it may cause milder disease or be less transmissible. It may be difficult for the spike protein to evolve to escape neutralizing antibodies while maintaining high affinity for ACE2.

The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target.

The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.

Comparison of Neutralizing Antibody Titers Elicited by mRNA and Adenoviral Vector Vaccine against SARS-CoV-2 Variants.

The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC 50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.

Lentiviral-Vector-Based Dendritic Cell Vaccine Synergizes with Checkpoint Blockade to Clear Chronic Viral Infection.

Dendritic cell vaccines are a promising strategy for the treatment of cancer and infectious diseases but have met with mixed success. We report on a lentiviral vector-based dendritic cell vaccine strategy that generates a cluster of differentiation 8 (CD8) T cell response that is much stronger than that achieved by standard peptide-pulsing approaches. The strategy was tested in the mouse lymphocytic choriomeningitis virus (LCMV) model. Bone marrow-derived dendritic cells from SAMHD1 knockout mice were transduced with a lentiviral vector expressing the GP33 major-histocompatibility-complex (MHC)-class-I-restricted peptide epitope and CD40 ligand (CD40L) and injected into wild-type mice. The mice were highly protected against acute and chronic variant CL-13 LCMVs, resulting in a 100-fold greater decrease than that achieved with peptide epitope-pulsed dendritic cells. Inclusion of an MHC-class-II-restricted epitope in the lentiviral vector further increased the CD8 T cell response and resulted in antigen-specific CD8 T cells that exhibited a phenotype associated with functional cytotoxic T cells. The vaccination synergized with checkpoint blockade to reduce the viral load of mice chronically infected with CL-13 to an undetectable level. The strategy improves upon current dendritic cell vaccine strategies; is applicable to the treatment of disease, including AIDS and cancer; and supports the utility of Vpx-containing vectors.

Lentiviral Vector-Based Dendritic Cell Vaccine Suppresses HIV Replication in Humanized Mice.

HIV-1-infected individuals are treated with lifelong antiretroviral drugs to control the infection. A means to strengthen the antiviral T cell response might allow them to control viral loads without antiretroviral drugs. We report the development of a lentiviral vector-based dendritic cell (DC) vaccine in which HIV-1 antigen is co-expressed with CD40 ligand (CD40L) and a soluble, high-affinity programmed cell death 1 (PD-1) dimer. CD40L activates the DCs, whereas PD-1 binds programmed death ligand 1 (PD-L1) to prevent checkpoint activation and strengthen the cytotoxic T lymphocyte (CTL) response. The injection of humanized mice with DCs transduced with vector expressing CD40L and the HIV-1 SL9 epitope induced antigen-specific T cell proliferation and memory differentiation. Upon HIV-1 challenge of vaccinated mice, viral load was suppressed by 2 logs for 6 weeks. Introduction of the soluble PD-1 dimer into a vector that expressed full-length HIV-1 proteins accelerated the antiviral response. The results support development of this approach as a therapeutic vaccine that might allow HIV-1-infected individuals to control virus replication without antiretroviral therapy.

DNA damage induces a SAMHD1-mediated block to the infection of macrophages by HIV-1.

Monocyte-derived macrophages (MDMs) are an important target for HIV-1 despite SAMHD1, a myeloid restriction factor for which HIV-1 lacks a counteracting accessory protein. The antiviral activity of SAMHD1 is modulated by phosphorylation of T592 by cyclin-dependent kinases (CDK). We show that treatment of MDMs with neocarzinostatin, a compound that introduces double strand breaks (DBS) in genomic DNA, results in the decrease of phosphorylated SAMHD1, activating its antiviral activity and blocking HIV-1 infection. The effect was specific for DSB as DNA damage induced by UV light irradiation did not affect SAMHD1 phosphorylation and did not block infection. The block to infection was at reverse transcription and was counteracted by Vpx, demonstrating that it was caused by SAMHD1. Neocarzinostatin treatment also activated an innate immune response that induced interferon-stimulated genes but this was not involved in the block to HIV-1 infection, as it was not relieved by an interferon-blocking antibody. In response to Neocarzinostatin-induced DNA damage, the level of the CDK inhibitor p21cip1 increased which could account for the decrease of phosphorylated SAMHD1. The results show that the susceptibility of MDMs to HIV-1 infection can be affected by stimuli that alter the phosphorylation state of SAMHD1, one of which is the DNA damage response.

A DNA sequence recognition loop on APOBEC3A controls substrate specificity.

APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.

SAMHD1 restricts HIV-1 replication and regulates interferon production in mouse myeloid cells.

SAMHD1 restricts the replication of HIV-1 and other retroviruses in human myeloid and resting CD4(+) T cells and that is counteracted in SIV and HIV-2 by the Vpx accessory protein. The protein is a phosphohydrolase that lowers the concentration of deoxynucleoside triphosphates (dNTP), blocking reverse transcription of the viral RNA genome. Polymorphisms in the gene encoding SAMHD1 are associated with Aicardi-Goutières Syndrome, a neurological disorder characterized by increased type-I interferon production. SAMHD1 is conserved in mammals but its role in restricting virus replication and controlling interferon production in non-primate species is not well understood. We show that SAMHD1 is catalytically active and expressed at high levels in mouse spleen, lymph nodes, thymus and lung. siRNA knock-down of SAMHD1 in bone marrow-derived macrophages increased their susceptibility to HIV-1 infection. shRNA knock-down of SAMHD1 in the murine monocytic cell-line RAW264.7 increased its susceptibility to HIV-1 and murine leukemia virus and increased the levels of the dNTP pool. In addition, SAMHD1 knock-down in RAW264.7 cells induced the production of type-I interferon and several interferon-stimulated genes, modeling the situation in Aicardi-Goutières Syndrome. Our findings suggest that the role of SAMHD1 in restricting viruses is conserved in the mouse. The RAW264.7 cell-line serves as a useful tool to study the antiviral and innate immune response functions of SAMHD1.

Restriction of diverse retroviruses by SAMHD1.

BACKGROUND: SAMHD1 is a triphosphohydrolase that restricts the replication of HIV-1 and SIV in myeloid cells. In macrophages and dendritic cells, SAMHD1 restricts virus replication by diminishing the deoxynucleotide triphosphate pool to a level below that which supports lentiviral reverse transcription. HIV-2 and related SIVs encode the accessory protein Vpx to induce the proteasomal degradation of SAMHD1 following virus entry. While SAMHD1 has been shown to restrict HIV-1 and SIV, the breadth of its restriction is not known and whether other viruses have a means to counteract the restriction has not been determined. RESULTS: We show that SAMHD1 restricts a wide array of divergent retroviruses, including the alpha, beta and gamma classes. Murine leukemia virus was restricted by SAMHD1 in macrophages yet removal of SAMHD1 did not alleviate the block to infection because of an additional block to viral nuclear import. Prototype foamy virus (PFV) and Human T cell leukemia virus type I (HTLV-1) were the only retroviruses tested that were not restricted by SAMHD1. PFV reverse transcribes predominantly prior to entry and thus is unaffected by the dNTP level in the target cell. It is possible that HTLV-1 has a mechanism to render the virus resistant to SAMHD1-mediated restriction. CONCLUSION: The results suggest that SAMHD1 has broad anti-retroviral activity against which most viruses have not found an escape.

SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.

SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.

Human immunodeficiency virus type 1 modified to package Simian immunodeficiency virus Vpx efficiently infects macrophages and dendritic cells.

The lentiviral accessory protein Vpx is thought to facilitate the infection of macrophages and dendritic cells by counteracting an unidentified host restriction factor. Although human immunodeficiency virus type 1 (HIV-1) does not encode Vpx, the accessory protein can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) in virus-like particles, dramatically enhancing their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. We localized the minimal Vpx packaging motif of simian immunodeficiency virus SIVmac(239) p6 to a 10-amino-acid motif and introduced this sequence into an infectious HIV-1 provirus. The chimeric virus packaged Vpx that was provided in trans and was substantially more infectious on MDDC and MDM than the wild-type virus. We further modified the virus by introducing the Vpx coding sequence in place of nef. The resulting virus produced Vpx and replicated efficiently in MDDC and MDM. The virus also induced a potent type I interferon response in MDDC. In a coculture system, the Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo, Vpx may facilitate transmission of the virus from dendritic cells to T cells. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells.

Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase.

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses.

APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons.

APOBEC3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and transposition of endogenous retroelements. One family member, APOBEC3A (hA3A), is an orphan, without any known antiviral activity. We show that hA3A is catalytically active and that it, but none of the other family members, potently inhibits replication of the parvovirus adeno-associated virus (AAV). hA3A was also a potent inhibitor of the endogenous LTR retroelements, MusD, IAP, and the non-LTR retroelement, LINE-1. Its function was dependent on the conserved amino acids of the hA3A active site, consistent with a role for cytidine deamination, although mutations in retroelement sequences were not found. These findings demonstrate the potent activity of hA3A, an APOBEC3 family member with no previously identified function. They also highlight the functional differences between APOBEC3 proteins. The APOBEC3 family members have distinct functions and may have evolved to resist various classes of genetic elements.