Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques.

Most human immunodeficiency virus type 1 (HIV-1) infections begin at mucosal surfaces. Providing a barrier of protection at these may assist in combating the earliest events in infection. Systemic immunization by intramuscular (i.m.) injection can drive mucosal immune responses, but there are data suggesting that mucosal immunization can better educate these mucosal immune responses. To test this, rhesus macaques were immunized with replicating single-cycle adenovirus (SC-Ad) vaccines expressing clade B HIV-1 gp160 by the intranasal (i.n.) and i.m. routes to compare mucosal and systemic routes of vaccination. SC-Ad vaccines generated significant circulating antibody titers against Env after a single i.m. immunization. Switching the route of second immunization with the same SC-Ad serotype allowed a significant boost in these antibody levels. When these animals were boosted with envelope protein, envelope-binding antibodies were amplified 100-fold, but qualitatively different immune responses were generated. Animals immunized by only the i.m. route had high peripheral T follicular helper (pTfh) cell counts in blood but low Tfh cell counts in lymph nodes. Conversely, animals immunized by the i.n. route had high Tfh cell counts in lymph nodes but low pTfh cell counts in the blood. Animals immunized by only the i.m. route had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity, whereas animals immunized by the mucosal i.n. route had higher ADCC antibody activity. When these Env-immunized animals were challenged rectally with simian-human immunodeficiency virus (SHIV) strain SF162P3 (SHIVSF162P3), they all became infected. However, mucosally SC-Ad-immunized animals had lower viral loads in their gastrointestinal tracts. These data suggest that there may be benefits in educating the immune system at mucosal sites during HIV vaccination.IMPORTANCE HIV-1 infections usually start at a mucosal surface after sexual contact. Creating a barrier of protection at these mucosal sites may be a good strategy for to protect against HIV-1 infections. While HIV-1 enters at mucosa, most vaccines are not delivered here. Most are instead injected into the muscle, a site well distant and functionally different than mucosal tissues. This study tested if delivering HIV vaccines at mucosa or in the muscle makes a difference in the quality, quantity, and location of immune responses against the virus. These data suggest that there are indeed advantages to educating the immune system at mucosal sites with an HIV-1 vaccine.

A Replicating Single-Cycle Adenovirus Vaccine Against Ebola Virus.

Recent West African Ebola virus (EBOV) epidemics have led to testing different anti-EBOV vaccines, including a replication-defective adenovirus (RD-Ad) vector (ChAd3-EBOV) and an infectious, replication-competent recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (rVSV-EBOV; also known as rVSV-ZEBOV). While RD-Ads elicit protection, when scaled up to human trials, the level of protection may be much lower than that of vaccines containing viruses that can replicate. Although a replication-competent Ad (RC-Ad) vaccine might generate a level of protection approximating that of rVSV, this infectious vector would also risk causing adenovirus disease. We recently described a "single-cycle" adenovirus (SC-Ad) vector that amplifies antigen genes like RC-Ad, but that avoids the risk of adenovirus infection. Here we have tested an SC-Ad6 vector expressing the glycoprotein (GP) from a 2014 EBOV strain in mice, hamsters, and rhesus macaques. We show that SC-Ad6-EBOV GP induces a high level of serum antibodies in all species and mediates significant protection against pseudo-challenge with rVSV-EBOV expressing luciferase in mice and hamsters. These data suggest that SC-Ad6-EBOV GP may be useful during future EBOV outbreaks.

Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses.

Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed "single-cycle" adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as "needle-free" mucosal vaccines. IMPORTANCE: Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold promise to be more potent vectors and vaccines than current RD-Ad vectors.

Comparison of systemic and mucosal immunization with replicating Single cycle Adenoviruses.

HIV-1 infections occur during sexual contact at mucosal surfaces. Vaccines need to provide mucosal barrier protection and stimulate systemic immune responses to control HIV spread. Most vaccines are delivered by systemic immunization via intramuscular (IM) injection route. While this can drive systemic and mucosal immune responses, there are data show that mucosal immunization may be superior at driving responses at mucosal barriers. To explore this question, we immunized mice with replicating single-cycle adenovirus (SC Ad) vaccines expressing clade B HIV-1 envelope (Env) by intramuscular (IM), intranasal (IN), or intravaginal (IVAG) routes to compare vaccine responses. SC-Ads generated significant antibodies against Env after only a single immunization by the IN route, but not the other routes. These animals were boosted by the same route or by the mucosal IVAG routes. IM and IN primed animals generated strong antibody responses regardless of the boosting route. In contrast, IVAG primed animals failed to generate robust antibodies whether they were boosted by the IVAG or IM routes. These data suggest there may be benefits in first educating the immune system at mucosal sites during HIV vaccination. IN and IM prime-boost were then compared in Syrian hamsters which support SC-Ad DNA replication. In this case, IN immunization again was the only route that generated significant Env antibodies after a single immunization. Following a boost by IN or IM routes, IN primed animals had significantly higher antibody responses than the IM primed animals. Env antibodies could still be detected one year after immunization, but only in animals that received at least one mucosal IN immunization. These data suggest that there is merit in vaccination by mucosal routes.

Retargeted and detargeted adenovirus for gene delivery to the muscle.

We previously selected muscle binding peptides 12.51 and 12.52 from "context-specific" phage display libraries for introduction into adenovirus (Ad) vectors. In this work, these peptides were inserted into the hypervariable region (HVR) 5 loop of the Ad5 hexon protein to display 720 peptides per virions. HVR-12.51 and 12.52 increased transduction of C2C12 cells up to 20-fold when compared to unmodified Ad5. 12.51 increased in vivo muscle transduction 2 to 7-fold over unmodified Ad after intramuscular injection in mice and hamsters. 12.52 did not increase muscle transduction. Notably, insertion of 12.51 into the hexon reduced liver transduction 80-fold when compared to unmodified Ad5 after intravenous injection. Increased muscle transduction in mice translated into increased immune responses after gene-based vaccination. These data suggest there are merits to retargeting and detargeting benefits to modifying the hexons of Ads with peptide ligands.