Harnessing anti-cytomegalovirus immunity for local immunotherapy against solid tumors.

Tumor infiltration by T cells profoundly affects cancer progression and responses to immunotherapy. However, the tumor immunosuppressive microenvironment can impair the induction, trafficking, and local activity of antitumor T cells. Here, we investigated whether intratumoral injection of virus-derived peptide epitopes could activate preexisting antiviral T cell responses locally and promote antitumor responses or antigen spreading. We focused on a mouse model of cytomegalovirus (CMV), a highly prevalent human infection that induces vigorous and durable T cell responses. Mice persistently infected with murine CMV (MCMV) were challenged with lung (TC-1), colon (MC-38), or melanoma (B16-F10) tumor cells. Intratumoral injection of MCMV-derived T cell epitopes triggered in situ and systemic expansion of their cognate, MCMV-specific CD4+ or CD8+ T cells. The MCMV CD8+ T cell epitopes injected alone provoked arrest of tumor growth and some durable remissions. Intratumoral injection of MCMV CD4+ T cell epitopes with polyinosinic acid:polycytidylic acid (pI:C) preferentially elicited tumor antigen-specific CD8+ T cells, promoted tumor clearance, and conferred long-term protection against tumor rechallenge. Notably, secondary proliferation of MCMV-specific CD8+ T cells correlated with better tumor control. Importantly, intratumoral injection of MCMV-derived CD8+ T cell-peptide epitopes alone or CD4+ T cell-peptide epitopes with pI:C induced potent adaptive and innate immune activation of the tumor microenvironment. Thus, CMV-derived peptide epitopes, delivered intratumorally, act as cytotoxic and immunotherapeutic agents to promote immediate tumor control and long-term antitumor immunity that could be used as a stand-alone therapy. The tumor antigen-agnostic nature of this approach makes it applicable across a broad range of solid tumors regardless of their origin.

HPV16 infection decreases vaccine-induced HPV16 antibody avidity: the CVT trial.

The HPV vaccine has shown sustained efficacy and consistent stabilization of antibody levels, even after a single dose. We defined the HPV16-VLP antibody avidity patterns over 11 years among women who received one- or three doses of the bivalent HPV vaccine in the Costa Rica HPV Vaccine Trial. Absolute HPV16 avidity was lower in women who received one compared to three doses, although the patterns were similar (increased in years 2 and 3 and remained stable over the remaining 8 years). HPV16 avidity among women who were HPV16-seropositive women at HPV vaccination, a marker of natural immune response to HPV16 infection, was significantly lower than those of HPV16-seronegative women, a difference that was more pronounced among one-dose recipients. No differences in HPV16 avidity were observed by HPV18 serostatus at vaccination, confirming the specificity of the findings. Importantly, point estimates for vaccine efficacy against incident, six-month persistent HPV16 infections was similar between women who were HPV16 seronegative and seropositive at the time of initial HPV vaccination for both one-dose and three-dose participants. It is therefore likely that this lower avidity level is still sufficient to enable antibody-mediated protection. It is encouraging for long-term HPV-vaccine protection that HPV16 antibody avidity was maintained for over a decade, even after a single dose.

A Prime-Pull-Amplify Vaccination Strategy To Maximize Induction of Circulating and Genital-Resident Intraepithelial CD8+ Memory T Cells.

Recent insight into the mechanisms of induction of tissue-resident memory (TRM) CD8+ T cells (CD8+ TRM) enables the development of novel vaccine strategies against sexually transmitted infections. To maximize both systemic and genital intraepithelial CD8+ T cells against vaccine Ags, we assessed combinations of i.m. and intravaginal routes in heterologous prime-boost immunization regimens with unrelated viral vectors. Only i.m. prime followed by intravaginal boost induced concomitant strong systemic and intraepithelial genital-resident CD8+ T cell responses. Intravaginal boost with vectors expressing vaccine Ags was far superior to intravaginal instillation of CXCR3 chemokine receptor ligands or TLR 3, 7, and 9 agonists to recruit and increase the pool of cervicovaginal CD8+ TRM Transient Ag presentation increased trafficking of cognate and bystander circulating activated, but not naive, CD8+ T cells into the genital tract and induced in situ proliferation and differentiation of cognate CD8+ TRM Secondary genital CD8+ TRM were induced in the absence of CD4+ T cell help and shared a similar TCR repertoire with systemic CD8+ T cells. This prime-pull-amplify approach elicited systemic and genital CD8+ T cell responses against high-risk human papillomavirus type 16 E7 oncoprotein and conferred CD8-mediated protection to a vaccinia virus genital challenge. These results underscore the importance of the delivery route of nonreplicating vectors in prime-boost immunization to shape the tissue distribution of CD8+ T cell responses. In this context, the importance of local Ag presentation to elicit genital CD8+ TRM provides a rationale to develop novel vaccines against sexually transmitted infections and to treat human papillomavirus neoplasia.

Low-Dose Methotrexate Prevents Primary and Secondary Humoral Immune Responses and Induces Immune Tolerance to a Recombinant Immunotoxin.

Recombinant immunotoxins (RITs) are chimeric proteins being developed for cancer treatment. They are composed of an Ab fragment that targets a cancer Ag and a cytotoxic portion of Pseudomonas exotoxin A. They are effective for patients with hematologic malignancies with defective immunity, but their efficacy against solid tumors is limited by anti-drug Ab (ADA) responses in immune-competent patients. Pre-existing Abs or immune memory owing to previous toxin exposure represent additional hurdles because they induce rapid and strong ADA responses. Here, we evaluated the efficacy of methotrexate (MTX) to prevent ADA formation against the mesothelin-targeting RIT LMB-100 in naive mice and in mice with pre-existing Abs. We found that low-dose MTX combined with LMB-100 completely suppressed the formation of ADAs in a dose- and frequency-dependent manner. Suppression of the immune response restored blood levels of LMB-100 and prevented its neutralization. Furthermore, combination of MTX with LMB-100 did not compromise the immune response against a second Ag given after stopping MTX, indicating specific immune tolerance. Adoptive transfer of splenocytes suppressed Ab responses to LMB-100 in recipient mice, indicating a durable immune tolerance. We conclude that combination of MTX and LMB-100 is effective at preventing immune responses in a durable, Ag-specific manner. We propose combining low-dose MTX in immune-competent cancer patients receiving RIT therapy to prevent immunogenicity. This approach could be applied to other immunogenic therapeutic agents and to proteins for which there is pre-existing immunity.

Tolerogenic nanoparticles restore the antitumor activity of recombinant immunotoxins by mitigating immunogenicity.

Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.

Adenovirus vector-based prime-boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins.

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV-infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus-specific antigens for vaccine-induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV-specific CD8+ T cells producing IFN-γ and TNF-α to the cervicovaginal tract. Importantly, the cervicovaginal CD8+ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue-resident memory CD8+ T cells. This prime-boost strategy targeting heterologous locations also induced circulating HPV-specific CD8+ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.

Moving forward with human papillomavirus immunotherapies.

Persistent human papillomavirus (HPV) is the primary etiologic agent of cervical cancer and causes a significant number of vulvar, penile, anal and oropharyngeal cancers. The development of highly effective HPV therapeutic vaccines is a reasonable goal given the recent advances in basic and applied immunology. A number of vaccine strategies designed to induce systemic T cell responses have been tested in clinical trials against high grade cervical or vulvar high grade neoplasia and cancers, but with limited success. In line with the emerging trend to focus more on the epithelial context of HPV infection and premalignant disease, it might be advantageous to develop vaccination strategies that promote trafficking of HPV-specific T cells into lesions and overcome the local immunosuppressive environment. The development of more biologically relevant animal models would improve the preclinical evaluation of therapeutic vaccine candidates. Finally, persistent infection and low grade lesions may prove to be easier targets for therapeutic vaccines, and these vaccines would likely be commercially viable in high income countries and valuable components in screen and treat programs in low resource settings.

Absence of both Sos-1 and Sos-2 in peripheral CD4(+) T cells leads to PI3K pathway activation and defects in migration.

Sos-1 and Sos-2 are ubiquitously expressed Ras-guanine exchange factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4(+) T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4(+) T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4(+) T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration.

Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

UNLABELLED: No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. IMPORTANCE: Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.

Mouse model of cervicovaginal papillomavirus infection.

Virtually all cervical cancers are caused by human papillomavirus infections. The efficient assembly of pseudovirus (PsV) particles incorporating a plasmid expressing a reporter gene has been an invaluable tool in the development of in vitro neutralization assays and in studies of the early mechanisms of viral entry in vitro. Here, we describe a mouse model of human papillomavirus PsV infection of the cervicovaginal epithelium that recapitulates the early events of papillomavirus infection in vivo.

Antibody to the gp120 V1/V2 loops and CD4+ and CD8+ T cell responses in protection from SIVmac251 vaginal acquisition and persistent viremia.

The human papillomavirus pseudovirions (HPV-PsVs) approach is an effective gene-delivery system that can prime or boost an immune response in the vaginal tract of nonhuman primates and mice. Intravaginal vaccination with HPV-PsVs expressing SIV genes, combined with an i.m. gp120 protein injection, induced humoral and cellular SIV-specific responses in macaques. Priming systemic immune responses with i.m. immunization with ALVAC-SIV vaccines, followed by intravaginal HPV-PsV-SIV/gp120 boosting, expanded and/or recruited T cells in the female genital tract. Using a stringent repeated low-dose intravaginal challenge with the highly pathogenic SIVmac251, we show that although these regimens did not demonstrate significant protection from virus acquisition, they provided control of viremia in a number of animals. High-avidity Ab responses to the envelope gp120 V1/V2 region correlated with delayed SIVmac251 acquisition, whereas virus levels in mucosal tissues were inversely correlated with antienvelope CD4(+) T cell responses. CD8(+) T cell depletion in animals with controlled viremia caused an increase in tissue virus load in some animals, suggesting a role for CD8(+) T cells in virus control. This study highlights the importance of CD8(+) cells and antienvelope CD4(+) T cells in curtailing virus replication and antienvelope V1/V2 Abs in preventing SIVmac251 acquisition.

BK polyomavirus genotypes represent distinct serotypes with distinct entry tropism.

BK polyomavirus (BKV) causes significant urinary tract pathogenesis in immunosuppressed individuals, including kidney and bone marrow transplant recipients. It is currently unclear whether BKV-neutralizing antibodies can moderate or prevent BKV disease. We developed reporter pseudoviruses based on seven divergent BKV isolates and performed neutralization assays on sera from healthy human subjects. The results demonstrate that BKV genotypes I, II, III, and IV are fully distinct serotypes. While nearly all healthy subjects had BKV genotype I-neutralizing antibodies, a majority of subjects did not detectably neutralize genotype III or IV. Surprisingly, BKV subgenotypes Ib1 and Ib2 can behave as fully distinct serotypes. This difference is governed by as few as two residues adjacent to the cellular glycan receptor-binding site on the virion surface. Serological analysis of mice given virus-like particle (VLP)-based BKV vaccines confirmed these findings. Mice administered a multivalent VLP vaccine showed high-titer serum antibody responses that potently cross-neutralized all tested BKV genotypes. Interestingly, each of the neutralization serotypes bound a distinct spectrum of cell surface receptors, suggesting a possible connection between escape from recognition by neutralizing antibodies and cellular attachment mechanisms. The finding implies that different BKV genotypes have different cellular tropisms and pathogenic potentials in vivo. Individuals who are infected with one BKV serotype may remain humorally vulnerable to other BKV serotypes after implementation of T cell immunosuppression. Thus, prevaccinating organ transplant recipients with a multivalent BKV VLP vaccine might reduce the risk of developing posttransplant BKV disease.

Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses.

The induction of persistent intraepithelial CD8+ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8+ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced 10-fold more cervicovaginal antigen-specific CD8+ T cells than priming alone. Antigen-specific T cell numbers decreased only 2-fold after 6 months. Most genital antigen-specific CD8+ T cells were intra- or subepithelial, expressed αE-integrin CD103, produced IFN-γ and TNF-α, and displayed in vivo cytotoxicity. Using a sphingosine-1-phosphate analog (FTY720), we found that the primed CD8+ T cells proliferated in the cervicovaginal mucosa upon HPV intravaginal boost. Intravaginal HPV prime/boost reduced cervicovaginal viral titers 1,000-fold after intravaginal challenge with vaccinia virus expressing the CD8 epitope M2. In contrast, intramuscular prime/boost with an adenovirus type 5 vector induced a higher level of systemic CD8+ T cells but failed to induce intraepithelial CD103+CD8+ T cells or protect against recombinant vaccinia vaginal challenge. Thus, HPV vectors are attractive gene-delivery platforms for inducing durable intraepithelial cervicovaginal CD8+ T cell responses by promoting local proliferation and retention of primed antigen-specific CD8+ T cells.

Neutralization serotyping of BK polyomavirus infection in kidney transplant recipients.

BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy.

Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA.

The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.

Mucosa-associated epithelial chemokine/CCL28 expression in the uterus attracts CCR10+ IgA plasma cells following mucosal vaccination via estrogen control.

Previous studies demonstrated cross talk between mucosal and reproductive organs during secretory IgA Ab induction. In this study, we aimed to clarify the underlying mechanisms of this cross talk. We found significantly higher titers of Ag-specific secretory IgA Ab in the vaginal wash after mucosal vaccination by both the intranasal (i.n.) and the intravaginal routes but not by the s.c. route. Interestingly, Ag-specific IgA Ab-secreting cells (ASCs) were found mainly in the uterus but not in the cervix and vaginal canal after i.n. vaccination. The fact that most Ag-specific IgA ASCs isolated from the uteri of vaccinated mice migrated toward mucosa-associated epithelial chemokine (MEC)/CCL28 suggests dominant expression of CCR10 on the IgA ASCs. Further, IgA ASCs in the uteri of vaccinated mice were reduced drastically in mice treated with neutralizing anti-MEC/CCL28 Ab. Most intriguingly, the female sex hormone estrogen directly regulated MEC/CCL28 expression and was augmented by i.n. vaccination with cholera toxin or stimulators for innate immunity. Further, blockage of estrogen function in the uterus by oral administration of the estrogen antagonist raloxifene significantly inhibited migration of Ag-specific IgA ASCs after i.n. vaccination with OVA plus cholera toxin. Taken together, these data strongly suggest that CCR10(+) IgA ASCs induced by mucosal vaccination via the i.n. route migrate into the uterus in a MEC/CCL28-dependent manner and that estrogen might have a crucial role in the protection against genital infection by regulating MEC/CCL28 expression in the uterus.

Characterization of monoclonal antibodies specific for the Merkel cell polyomavirus capsid.

Merkel cell polyomavirus (MCV) has been implicated as a causative agent in Merkel cell carcinoma. Robust polyclonal antibody responses against MCV have been documented in human subjects, but monoclonal antibodies (mAbs) specific for the VP1 capsid protein have not yet been characterized. We generated 12 mAbs capable of binding recombinant MCV virus-like particles. The use of a short immunogenic priming schedule was important for production of the mAbs. Ten of the 12 mAbs were highly effective for immunofluorescent staining of cells expressing capsid proteins. An overlapping set of 10 mAbs were able to neutralize the infectivity of MCV-based reporter vectors, with 50% effective doses in the low picomolar range. Three mAbs interfered with the binding of MCV virus-like particles to cells. This panel of anti-capsid antibodies should provide a useful set of tools for the study of MCV.

Sublingual immunization with an HIV subunit vaccine induces antibodies and cytotoxic T cells in the mouse female genital tract.

A vaccine against heterosexual transmission by human immunodeficiency virus (HIV) should generate cytotoxic and antibody responses in the female genital tract and in extra-genital organs. We report that sublingual immunization with HIV-1 gp41 and a reverse transcriptase polypeptide coupled to the cholera toxin B subunit (CTB) induced gp41-specific IgA antibodies and antibody-secreting cells, as well as reverse transcriptase-specific CD8 T cells in the genital mucosa, contrary to intradermal immunization. Conjugation of the reverse transcriptase peptide to CTB favored its cross-presentation by human dendritic cells to a T cell line from an HIV(+) patient. Sublingual vaccination could represent a promising vaccine strategy against heterosexual transmission of HIV-1.