First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers.

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 105 to 2.5 × 108 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4+ and CD8+ T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 105 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies.

Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses.

An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

Heterologous prime-boost immunizations with a virosomal and an alphavirus replicon vaccine.

Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of inducing strong CTL responses in homologous prime-boost protocols. Here, we demonstrate that a heterologous prime-boost with recombinant Semliki Forest virus (rSFV) encoding a fusion protein of E6 and E7 of human papillomavirus (HPV) type 16 and virosomes containing the HPV16 E7 protein resulted in higher numbers of antigen-specific CTL in mice than homologous protocols. Evasion of vector-specific immunity appeared to play a role in establishing these high frequencies, as coinduction of vector-specific responses during the prime immunization reduced the frequency of antigen-specific CTL after a heterologous booster. However, the high numbers of CTL initially primed by the heterologous protocols did not correlate with enhanced responsiveness to in vitro antigenic stimulation, nor in improved cytolytic activity or antitumor responses in vivo compared to a homologous protocol with rSFV. This lack of correlation could not be explained by changes in numbers of regulatory T cells. However, we observed differences in the frequencies of T cell subsets within the E7-specific CD8(+) T cell population, e.g. higher frequencies of central memory T cells upon homologous immunizations compared to heterologous immunizations. The induction of central memory T cells is crucial for a cancer vaccine as these cells are known to rapidly expand upon recall stimulation. This study demonstrates that the strongly increased number of antigen-specific CTL as induced by heterologous prime-boost immunizations, often used as a proof for the enhanced efficacy of such regimes, does not necessarily equal superior functional antitumor responses.

Superior therapeutic efficacy of alphavirus-mediated immunization against human papilloma virus type 16 antigens in a murine tumour model: effects of the route of immunization.

In our efforts to develop a strong, effective immune response against cervical carcinoma and premalignant disease, we study the use of recombinant Semliki Forest virus (SFV) encoding the oncoproteins E6 and E7 from high-risk human papilloma viruses (HPVs). Optimal immunization conditions are required for immunotherapeutic treatment of cervical cancer as it has been postulated that cervical cancer patients are immune-suppressed and/or immunologically tolerant for HPV. We previously generated an optimized construct encoding a fusion protein of HPV16 E6 and E7 and a translational enhancer (enhE6,7). Immunization of mice with SFV-enhE6,7 was shown to induce cytoxic T cell (CTL) responses and resulted in the eradication of established tumours. We now demonstrate, using HPV16-specific MHC class I tetramers, that high pCTL frequencies can be induced. However, this induction is strongly influenced by the route of immunization applied. Whilst in bulk CTL assays, requiring in vitro restimulation, CTL activity can be observed upon s.c., i.p., i.v. and i.m. immunization, detectable pCTL frequencies, without in vitro restimulation, are only induced upon i.m. and i.v. immunization. The route of immunization also strongly influences the dose of viral vector needed to induce CTLs and tumour therapy. As few as 5x10(4) SFV-enhE6,7, primed and boosted i.v., are needed to eradicate tumours in six out of seven mice treated. Furthermore, exponentially growing tumours of approximately 500 mm3 in size were seen to completely resolve and even tumours as large as 1500 mm3 decreased to one-third of their size. Apart from this potency, SFV vectors can safely be used for the expression of oncoproteins such as E6 and E7, since the viral RNA is not integrated in the host genome. Thus SFV-enhE6,7 meets with the criteria that a vaccine against cervical cancer should be safe and induce a very strong, long-lasting CTL response, strong enough to eradicate existing tumours.

Role of regulatory T-cells in immunization strategies involving a recombinant alphavirus vector system.

BACKGROUND: Regulatory T-cells (Treg) hamper immune responses elicited by cancer vaccines. Therefore, depletion of Treg is being used to improve the outcome of vaccinations. METHODS: We studied whether an alphavirus vector-based immunotherapeutic vaccine changes the number and/or activity of Treg and if Treg depletion improves the efficacy of this vaccine against tumours. The vaccine is based on a Semliki Forest virus (SFV). The recombinant SFV replicon particles encode a fusion protein of E6 and E7 from human papillomavirus (HPV) type 16 (SFVeE6,7). RESULTS: We demonstrated that SFVeE6,7 immunization did not change Treg levels and their suppressive activity. Depletion of Treg in mice, using the novel anti-folate receptor 4 antibody, did not enhance the immune response induced by SFVeE6,7 immunization. Both the priming and the proliferation phases of the HPV-specific response elicited with SFVeE6,7 were not affected by the immune-suppressive activity of Treg. Moreover, Treg depletion did not improve the therapeutic antitumour response of SFVeE6,7 in a murine tumour model. CONCLUSIONS: The efficacy of the SFVeE6,7 vaccine was not hampered by Treg. Therefore, SFVeE6,7 seems a very promising candidate for the treatment of HPV-induced disease, as it may not require additional immune interventions to modulate Treg activity.

Role of T cell competition in the induction of cytotoxic T lymphocyte activity during viral vector-based immunization regimens.

T cell competition between antigen- and vector-specific T cells may determine the outcome of viral vector-based immunization regimens, as we previously proposed. Here, we unravelled the interplay between antigen- and vector-specific immunity, using recombinant Semliki Forest virus (rSFV). Priming of mice with rSFV, encoding LacZ (SFVLacZ), or with empty rSFV strongly suppressed subsequent induction of ovalbumin or Human Papilloma virus (HPV) E6/E7-specific CTL activity by a booster with SFVeOVA or SFVeE6,7, respectively. Yet, priming with irradiated, i.e. replication-defective, SFVLacZ did not affect subsequent CTL induction, indicating that the interfering vector-specific immunity was directed against the viral replicase. However, immune responses against the strongly immunogenic nucleoprotein of influenza virus encoded by SFV were less severely affected by priming with SFVLacZ. Thus the outcome of heterologous prime-boost immunizations appears to depend on the immunogenicity of the respective antigens.

Augmentation of alphavirus vector-induced human papilloma virus-specific immune and anti-tumour responses by co-expression of interleukin-12.

To enhance the efficacy of a therapeutic immunisation strategy against human papillomavirus-induced cervical cancer we evaluated the adjuvant effect of interleukin-12 (IL12) expressed by a Semliki Forest virus vector (SFV) in mice. Depending on the dose and schedule, SFV-IL12 stimulated antigen-specific CTL responses elicited upon immunisation with recombinant SFV expressing HPV16-E6E7 (SFVeE6,7). SFVeE6,7-CTL and anti-tumour activity were enhanced by a low dose of SFV-IL12 to the prime immunisation. Using higher dosages these activities were reduced. Addition of SFV-IL12 to the booster immunisation further reduced the efficacy of the SFVeE6,7 immunisation. In transgenic mice, tolerant for HPV16-E6E7, SFV-IL12 also stimulated SFVeE6,7-induced CTL responses. In conclusion, SFV-IL12 can enhance antigen-specific immune responses. Yet, prudence is called for when considering co-administration of SFV-IL12 to a vaccine, as the enhancement of cell-mediated immune responses greatly depends on dosage and schedule.

The effect of pre-existing immunity on the capacity of influenza virosomes to induce cytotoxic T lymphocyte activity.

Protein antigens encapsulated in virosomes generated from influenza virus can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. In the present study we determined, in a murine model system, whether pre-existing immunity against influenza virus hampers the induction of a CTL response. CTL induction was only slightly reduced by pre-injection of influenza virus-specific antibodies or pre-exposure to influenza virus. Both pretreatments resulted in the same level of reduction, suggesting that virus-specific antibodies rather than T cell responses account for the reduction. Furthermore, a booster immunization enhanced CTL activation, indicating that virosome-specific immunity induced by a prime immunization does not hamper the booster effect. In conclusion, CTL induction against virosome-encapsulated protein antigens is not significantly inhibited by pre-existing humoral or cellular immunity against influenza virus.

Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7.

Previously, we described the efficacy of immunisation with recombinant Semliki Forest virus (SFV), expressing the human papillomavirus 16 (HPV) oncoproteins E6 and E7, in inducing HPV-specific CTLs and anti-tumour responses. Recently, we developed a novel recombinant SFV construct encoding a relatively stable fusion protein of HPV16 E6 and E7 under control of a translational enhancer derived from the SFV capsid protein. In the present study we demonstrate that immunisation of tumour-bearing mice with this improved vector results in the regression and complete elimination of established tumours. We furthermore demonstrate that a long-term high level of CTL activity, up to 340 days, accompanies the anti-tumour response. Thus, immunisation with recombinant SFV particles encoding increased levels of a fusion protein of HPV16 E6 and E7 efficiently induces CTL activity and CTL memory resulting in a potent therapeutic anti-tumour effect.