​​​CEA-expressing cancer (eg, colon cancer)
  - breaking tolerance & enhanced survival potential

  - CEA antibody responses

In collaboration with Duke University Medical Center and with support from NCI, a Phase I / II study (open-label, dose-escalation study) was conducted to evaluate the safety and immunogenicity of carcinoembryonic antigen (CEA (6D)) - expressing virus-like replicon particle (VRP) vaccine (AVX701) in patients with CEA-expressing malignancies. The subjects with advanced or metastatic (Stage IV) CEA-expressing malignancies were treated with one of three escalating doses or CEA-expressing VRP. in thesis subjects, AVX701 was administered by intramuscular (IM) injection every 3 weeks for a minimum of four immunizations, with additional doses patient's without progressive disease every 3 months. 

A total of 28 subjects were treated in the dose-ranging part of the study and no safety or toxicity issues were identified, confirming the favorable safety profile of this intervention seen in other studies (Morse et al. 2010 JCI). Following repeated administration, AVX701 was shown to be effective in eliciting CEA -specific T cell and antibody response even in the presence of anti-vector neutralizing antibodies and elevated Treg levels. The study results suggested that patients with CEA-specific T cell responses exhibited longer overall survival.

A study enrolling patients with Stage III cancer was subsequently initiated and closed to new participants in  2017. Patients (12) were administered the candidate vaccine intramuscularly every three weeks for four administrations. VRP-CEA activated CEA-specific antibodies with above-baseline titers were seen in all. The titer, in Stage IV, of 80 rose to almost 500 in Stage III patients. A median follow up of 60 months showed that all were alive, three parients showed recurrent disease. The anti-tumor immune response was demonstrated by a reduction in Tregs and an increase in the antigen-specific effector T cells.



VRP Vaccine
Ga(myr-) (AVX101)

Clinical Results 
The two component alphavaccine (AVX601) expressing CMV gB or a pp65/IE1 fusion protein was evaluated in a randomized, double-blind Phase 1 clinical trial in CMV-seronegative subjects ( Bernstein et al. 2009 Vaccine 28 ). Forty subjects received a lower dose (LD) or higher dose (HD) of vaccine or placebo by intramuscular or subcutaneous injection at Weeks 0, 8 and 24. The vaccine was well tolerated, with mild to moderate local reactogenicity, minimal systemic reactogenicity, and no clinically important changes in laboratory parameters. 

All vaccine recipients developed ex vivo, direct IFN-gamma_ELISPOT responses to CMV antigens. Polyfunctional CD4+ and CD8+ T cell responses were detected by polychromatic flow cytometry. These analyses demonstrated that a substantial proportion of the CMV-specific T cell responses had polyfunctional phenotypes thought to be important for protective immune responses. CMV antigen-specific CD4 + and CD8 + effector T cells producing multiple cytokines in response to CMV peptide stimulation were induced in all subjects immunized with AVX601. CD8 + T cells stimulated with pp65 produced the highest percentage of cytokine-positive cells. CD4 + T cells exhibited responses representing all of the possible 7 cytokine profiles with a high proportion of IFN γ + TNF α + IL-2 + triple producers. The absence of a detectable IL-4 resp onse by CD4 + T cells after stimulation with each of the three CMV peptide pools is consistent with a Th1- biased response to AVX601. Polyfunctional T cells were detectable after one dose, sometimes increased in frequency after subsequent doses, and were still present 6 months after the third dose of vaccine. These data show that AVX601 elicited a highly desirable, complex cytokine response in both CD8 + and CD4 + T cell subsets to the three CMV proteins expressed by AV X601. 

AVX601 vaccine induced neutralizing antibodies to CMV, presu mably directed at gB, in all recipients of high dose vaccine. Titers remained read ily detectable in 53% and 75% of low dose and high dose subjects,resepectively, at six months post 3rd dose. 

In sum, AVX601 was safe and induced both polyfunctional T cell responses against three CMV antigens and neutralizing antibodies to gB that are important targets for protective immunity.

Vaccine for HIV-1 
AlphaVax received multi-year grant support to develop a candidate alphavaccine for HIV-1. This support came initially from the International AIDS Vaccine Initiative (IAVI) and subsequently from the US government, through the Division of AIDS at the National Institutes of Health. ​

a Vaccine was administered at SW 0, 4 and 24. The ELISPOT a nd ELISA results are from SW6 and SW26 PBMC and serum samples.

Cancer Immunotherapy

The alphavaccine vector system is considered to be unusually well-suited for tumor immunotherapy because of its ability to generate a comprehensive immune response with both antibodies and T cells that specifically target tumor-associated antigens, and an apparent ability to sustain immunologic activity over repeated use despite the presence of an immune response to the vector. Both cytotoxic T lymphocytes (CTL) and antibodies (e.g., Herceptin P ® ) are recognized as key effector mechanisms in anti-cancer immunity. However, most tumor -associated antigens are “self” proteins, and if active cancer immunotherapy is to be effective, a vaccine must be able to break the immunological tolerance to self antigens. In preclinical studies, alphavaccines have been demonstrated to break tolerance in mice transgenic for neu, the rat homolog of HER2, as well as in rats expressing rat HER2/ neu. As part of a program project grant from NCI for development of an immunotherapeutic vaccine for colon cancer, pre-clinical studies demonstrated that tolerance to self antigen was broken in mice transgenic for carcinoembryonic antigen (CEA) by alphavaccines expressing CEA. These results provided the support to advance this vaccine into clinical testing. 

Vaccine for Cytomegalovirus (CMV)

Protective immunity to CMV involves both humoral and cellular mechanisms. Therefore an effective vaccine will need to stimulate both neutralizing antibody and T cell responses to the vaccine expressed viral antigens. AlphaVax designed its CMV alphavaccine candidate to express three different CMV proteins (AVX601). Two proteins, pp65 and I E1, are the principal targets for cellular immune responses in CMV seropositive individuals, and a third, glycoprotein gB, is the principal target for neutralizing antibody responses. Extensive preclinical data demonstrated the ability of alphavaccines expressing these proteins to elicit a balanced immune response to the three CMV proteins in animal models. 

Clinical Results
Two clinical trials (HVTN 040 and HVTN 059) evaluating the safety and immunogenicity of a similar alphavirus replicon particle vaccine that expressed non-myristoylated Gag, HIV Gag VRP vaccine (AVX101), were conducted under BB-IND 10171 in the United States (US) and southern Africa (SA), using dosage l evels of 1 x 10 4 and 1 x 10 5 IU in study 040 and 1 x 10 5 , 1 x 10 6 , 1 x 10 7 and 1 x 10 8 IU in study 059. A total of 132 HIV seronegative participants were enrolled (24 at the 1 x 10 4 IU dosage level, 48 at the 1 x 10 5 IU dosage level, 24 each at the 1 x 10 6 and 1 x 10 7 IU dosage levels, and 12 at the 1 x 10 8 IU dosage level). Review of safety data showed the vaccine was well tolerated, with local and systemic reactogenicity generally reported as none or mild to moderate. Two systemic reactogenicity events (one fatigue and one headache) were reported as severe, each of which resolved within 1 day. No serious safety concerns were identified. 

HVTN 059 was a randomized, placebo-controlled, double-blind phase I trial that evaluated the safety and immunogenicity of an alphavirus replicon HIV-1 subtype C Gag vaccine (AVX 101) in healthy, HIV-1 uninfected adults at doses of 10 5 to 10 8 infectious units (IU) given subcutaneously three times at 0, 1, and 3 months. The Gag expressed in this vaccine construct was non-myristoylated and therefore the expressed protein could not assemble into Gag-derived virus-like particles (VLPs). The immunogenicity results for the participants in the HVTN 059 trial demonstrated a dose-dependent anti-Gag antibody response as measured by Gag-specific (p55) ELISA. After three doses of vaccine, the antibody response in the active vaccine recipients showed a response rate of 100% at the 10 8 IU dosage level and 80% at the 10 7 IU dosage level. These response rates were markedly better than published results with ALVAC, DNA or MVA vectors expressing Gag. A positive cytotoxic T lymphocyte (CTL) response was seen in nine participants (three in the 10 8 IU group and six at lower dosage levels) and a positive gamma interferon enzyme-linked immunospot (IFN- γ ELISPOT) response was seen in two participants (one each in the 10 7 and 10 8 IU groups). There was also a dose-related cellular immune response as measured by an intracellular cytokine staining (ICS) a ssay. Two weeks after the third dose of vaccine, CD4 T cells secreting both interleukin 2 (IL-2) and IFN- γ were detected in 0% and 22% of subjects at the two lower dosage levels and 50% and 30% of subjects at the two higher dosage levels. The magnitude and frequency of the Gag-specific T cell responses measured in the AVX101 vaccinated subjects contrasted significantly with those measured in preclinical murine immunogenicity studies in which AVX101 was shown to elicit robust T cell responses as measured by ex-vivo, direct IFN- γ ELISPOT. Subsequent immunogenicity studies in non-human primates (NHP) also showed that AVX101 induced poor T cell responses while stimulating anti-Gag antibody responses. The NHP immunogenicity results with AVX101 mimicked those from HVTN 059. However, in a separate study in NHP, a VRP vaccine that expressed the myristoylated form of Gag from a clade B isolate of HIV-1 was shown to elicit robust Gag-specific antibody and T cell responses. Comparison of the NHP immunogenicity results using the myristoylated form of Gag with those from AVX101 vaccinated NHPs suggested that expression of myristoylated Gag and subsequent assembly of Gag-derived VLPs was required to induce robust Gag-specific T cell responses in primates. 

To test this hypothesis, the immunogenicity of VRP vaccines containing myristoylated Gag (myr+) and non-myristoylated Gag (myr-) (AVX 101) were compared in NHP (Chinese rhesus macaques). As shown in the table below, the peak Gag-specific T cell responses as measured by IFN- γ ELISPOT after the 2nd and 3rd doses of vaccine were 4 to 5 fold greater in Gag (myr+) vaccinated animals than those measured in G ag (myr-) (AVX101) vaccinated monkeys. All five Gag (myr+) vaccinated animals showed a positive Gag response by 4 weeks post-priming dose and remained positive at each of the time points through study’s end. In contrast, only 4 of 5 AVX101 vaccinated monkeys showed positive responses at one or two time points after the second dose. One of 5 did not have a measurable T cell response. Irrespective of the vaccine received, all animals mounted anti-Gag antibody responses after two doses of vaccine and in contrast to the vaccine specific differences in T cell responses, the Gag-specific ELISA titers induced by either vaccine were of similar magnitude (Table 1). 

In two separate groups, a total of 10 macaques also received 3 doses of HIV gp160 VRP. All 10 animals developed anti-gp120 antibodies by SW6 (2 weeks af ter the 2 nd dose) and those antibody levels were boosted following a 3rd dose of vaccine (Table 1). All 10 animals had measurable gp160 specific T cell responses at one or more time points across the study. Low level HIV neutralizing antibody activity was detected after the 3rd dose of gp160 VRP in serum from all gp160 VRP immunized animals (data not shown). HIV- 1 gp160-specific T cell responses were also measured by IFN- γ ELISPOT. The peak T cell responses were observed at 2 weeks after the 2nd dose of vaccine (Table 1). 

The results from this comparative immunogenicity study were e ncouraging and warrant, at a minimum, the clinical evaluation of the Gag(myr+) VRP vaccine to determine in human subjects whether a VRP vaccine expressing Gag (myr+) would e licit more frequent and higher magnitude Gag-specific T-cell responses.

Table 1. Immunization with AVX202 induced robust cellular and humoral immune responses in Chinese rhesus macaques.


AlphaVax has advanced seven different alphavaccines into eight Phase I / II clinical trials, and the results available for some of these trials are summarized below. The seven vaccine candidates were split between three in cancer (and four associated clinical trials) and four tested as anti-infectives in Phase I trials. Cumulatively, thesis trials have enrolled approximately 460 subjects and, to date, the results demonstrate a favorable safety profile for the vaccine platform as all candidate vaccines have been shown to be very well tolerated, with only mild to moderate local reactogenicity; and no safety issues have been observed. From the immunogenicity assessments conducted to date for CMV, influenza and HIV measurable immune responses, humoral, cellular or both, have been observed in all trial subjects who received two or three inoculations of the highest dosage level tested. In addition, from the initial cancer immunotherapy trial, the results suggest that the alphavaccine vector can break tolerance to a tumor-associated antigen, appears to impact positively survival and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression. 

​​​​​​HER2-expressing cancer (breast cancer)

  - positive T-cell responses

A study of 22 patients, either with advanced or metastatic breast cancer was completed. All of the patients had HER2-expressing malignancy. A full analysis and discussion of this study was published in January 2019 (http://clincancerres.aacrjournals.org/content/early/2019/01/11/1078-0432.CCR-18-3102). Entitled "Vaccine-induced memory CD8+ T cells provide clinical benefit in HER2 expressing breast cancer: a mouse to human translational study", the report concludes that VRP-HER2 increased HER2-specific memory CD8 T cells, had anti-tumor effects, and that the expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased progression-free survival. A Phase II trial comparing the vaccine candidate against pembrolizumab and against a combination of the latter two is in progress.

Clinical Results – Seasonal Influenza Vaccine 
AlphaVax has carried out two clinical studies of a pr ototype seasonal influenza A alphavaccine following promising immunogenicity and protection resul ts from extensive preclinical studies in animal models. 

The first clinical trial was a placebo-controlled, ra ndomized, double-blind study in 216 healthy volunteers (18-40 years of age), which evaluated the saf ety and humoral and cellular immune responses after one or two inoculations. The alphavaccine expressed the H3 HA gene from the A/Wyoming H3N2 strain of influenza virus. The vaccine was administered either subcutaneously or intramuscularly at two dosage levels, and was found to be safe and well tolerated irrespective of the route or the dose given. Both antibody and T cell responses were efficiently stimulated and persisted for the duration of the four-month study. Among volunteers with pre-vaccination influenza antibody titers (which are measured by a hemagglutination inhibition, or HI, assay) that were below levels thought to be protective, 77% and 80% of these individuals receiving a single low or high dose, respectively, responded with protective HI antibody titers. A second immunization in these individuals increased seroprotective responses to 86% for both dosage levels. A rapid and dose-dependent T cell response (measured by antigen-specific interferon (IFN)-gamma ELISPOT assay) was also observed and remained significantly elevated for at least four months. A second immunization extended the duration, but not the magnitude, of these T cell responses. For both anti body and cellular responses, there was no significant difference observed between subcutaneous and intr amuscular vaccinations. 

These results were encouraging and demonstrated that the a lphavaccine vector vaccine elicited a balanced immune response consisting of both hum oral and cellular responses. Comparable T-cell responses are typically not seen in adult populations with existing licensed influenza vaccines. HI antibody responses are known to correlate with protection against influenza infection and reduction of clinical disease, and influenza specific T cell responses are believed to function in eventual clearance of the virus from infected individuals. 

The same alphavaccine was also tested in a Phase I t rial involving 28 healthy adults, 65 years of age or older. The trial was a placebo-controlled, randomized, double-blind study that evaluated responses after administration of two doses of vaccine given at a single dosage level. The vaccine was safe and very well tolerated in this group of healthy, ambulatory elderly subjects, paralleling the experience in young adults. At four weeks after the 2nd dose, ten of twenty vaccine recipients had a 4-fold or greater rise in HI antibody titer compared to baseline. A significant increase in T cell responses was also observed in those receiving vaccine at this time point. None of the eight placebo subjects had measurable increases in antibody or T cell responses. The HI antibody responses measured in the subjects receiving vaccine exceeded the current regulatory approval guidelines for seroconversion in this age group. 

These results in the elderly demonstrate the potential of the AlphaVax platform for the development of alphavaccines for the elderly to target viral diseases, such as pandemic and seasonal influenza, respiratory syncytial virus, and varicella zoster.

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Vaccines for Influenza 

Preclinical Results 
Alphavaccines expressing influenza hemagglutinin (HA) have be en shown to protect mice, chickens and ferrets against influenza challenge, including strains of the highly virulent, potentially pandemic H5N1 avian strain in chickens. In mice, the immunogenicity of an alphavaccine expressing HA from influenza A/Vietnam/1203/2004 (H5N 1) virus is significantly greater than the immunogenicity of an H5 recombinant protein from the same virus strain. The immunogenicity of an alphavaccine expressing the HA from the sw ine-origin 2009 H1N1 influenza virus induces HI antibody levels exceeding those required for protection in mice, pigs, and nonhuman primates.