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

Abstract

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 sc, ip, iv and im immunization, detectable pCTL frequencies, without in vitro restimulation, are only induced upon im and iv immunization. The route of immunization also strongly influences the dose of viral vector needed to induce CTLs and tumour therapy. As few as 5x104 SFV-enhE6,7, primed and boosted iv, 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.

Heterologous Prime-Boost Immunizations with a Virosomal and an Alphavirus Replicon Vaccine

“…Mice were primed and boosted 14 days later with rSFV expressing a fusion protein of HPV 16 E6 and E7 (SFVeE6,7) or E7-containing virosomes (E7-virosomes) in homologous and heterologous combinations. Based on previous studies, ,, doses that induce strong CTL responses in homologous prime-boost protocols were used: 10 6 SFVeE6,7 particles and 50 nmol of virosomal phospholipids (i.e., ∼2.5 μg of E7 protein) respectively. Ten days after the booster immunization, the mice were sacrificed and spleens were collected.…”

“…Recombinant SFV was produced as previously described. , In brief, the plasmids pSFV3 and pSFV3 containing the β-Gal sequence (pSFV-β-gal) were purchased from Life Technologies . The plasmid pSFV-Helper 2 was kindly provided by Dr. Peter Liljeström, Stockholm, Sweden .…”

“…We have developed immunization strategies based on a virosomal antigen delivery system , or based on the recombinant Semliki Forest virus (rSFV) vector system. , E7 protein containing virosomes and rSFV encoding a fusion protein of HPV16 E6 and E7 have been shown to be very effective in inducing CTL responses against HPV16 E6- and E7-expressing cells. − Virosomes, in our studies derived from influenza virus, are reconstituted virus envelopes that retain the cell entry properties of the native influenza virus . These virosomes can be taken up by professional antigen presenting cells (APC) via receptor-mediated endocytosis.…”

Cancer vaccine strategies using self-replicating RNA viral platforms

“…2012 58 SFV P1A Mouse VRP Mastocytoma Anti-tumor immunity in vivo Ni et al, CDP, 2004 39 SFV P1A/IL-12 Mouse VRP Mastocytoma Tumor regression with activation of immune memory with rSFV-P1A + rSFV-IL-12 combo treatment P. Colmenero et al, Int. Journal of Cancer 2002 57 VEE E7 Virus VRP Cervical Cancer Anti-tumor protection for E6/E7 expressing cells Daemen et al, Gene Therapy, 2000 42 VEE E7 Virus VRP Cervical Cancer Anti-tumor immunity in vivo (CD8 + Dependent) Velders et al, Cancer Res., 2001 46 SFV E6 + E7 Virus VRP Cervical Cancer Enhanced in vivo protection against tumor challenge with fusion E6/E7 Daemen et al, GT 2002 41 SFV E6 + E7 Virus VRP Cervical Cancer In vivo protection against established tumors (long term) Daemen et al, Vaccine 2003 40 SFV E6 + E7 Virus VRP Cervical Cancer Efficacy of VRP tattoo delivery van de wall et al, Vaccines 2015 44 SFV E6 + E7 Virus VRP Cervical Cancer Superior anti-tumor responses from IM and IV administration routes Daemen et al, Antiviral Therapy 2004 43 SFV E6 + E7 Virus VRP Cervical Cancer VRP allows for prime-boosting of responses in comparison to Ad vectors Riezebos-Brilman et al, Gene Therapy ...…”

“…Vaccination using E6/E7 VRPs stimulated potent E6- and E7-specific T-cell immunity that translated into rejection of E6/E7 tumor challenge and suppressed the growth of established E6/E7 expressing tumors [ 40 – 42 ]. These vaccines proved to be most potent when delivered by intramuscular or intravenous routes (compared to subcutaneous and intradermal) and conferred long-term immunity against tumor challenge [ 40 , 43 ]. Subsequent studies also determined that administration by tattoo yielded comparable responses to intramuscular injection [ 44 ].…”