The immunological impact of adenovirus early genes on vaccine-induced responses in mice and nonhuman primates.

Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Ad with a deletion in early region 3 (ΔE3) provokes a stronger immune response than Ad with deletions in early regions 1 and E3 (ΔE1/ΔE3). The ΔE1/ΔE3 Ads are more popular because they can carry a larger transgene and because of the deleted E1 (E1A and E1B), are perceived safer for clinical use. Ad with a deletion in E1B55K (ΔE1B55K) has been in phase III clinical trials for use in cancer therapy in the US and has been approved for use in head and neck tumor therapy in China, demonstrating that Ad containing E1A are safe for clinical use. We have shown previously that ΔE1B55K Ad, even while promoting lower levels of an inserted transgene, promoted similar levels of transgene-specific immune responses as a ΔE3 Ad. Products of the Ad early region 4 (E4) limit the ability of cells to mount an innate immune response. Using this knowledge, we deleted the Ad E4 open reading frames 1-4 (E4orf1-4) from the ΔE1B55K Ad. Here, we show that innate cytokine network genes are elevated in the ΔE4 Ad-infected cells beyond that of ΔE3 Ad-infected cells. Further, in immunized mice the IgG2a subclass was favored as was the IgG1 subclass in immunized nonhuman primates. Thus, Ad E4 impacts immune responses in cells, in immunized mice, and immunized nonhuman primates. These Ad may offer advantages that are beneficial for clinical use.Importance: Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Here we provide evidence in cells, mice, and nonhuman primates supporting the notion that Ad early gene-products limit specific immune responses. Ad constructed with deletions in early genes and expressing HIV envelope protein was shown to induce greater HIV-specific cellular immune responses and higher titer antibodies compared to the parental Ad with the early genes. In addition to eliciting enhanced immunity, the deleted Ad possesses more space for insertion of additional or larger transgenes needed for targeting other infectious agents or cancers.

A Prime/Boost Vaccine Regimen Alters the Rectal Microbiome and Impacts Immune Responses and Viremia Control Post-Simian Immunodeficiency Virus Infection in Male and Female Rhesus Macaques.

An efficacious human immunodeficiency virus (HIV) vaccine will likely require induction of both mucosal and systemic immune responses. We compared the immunogenicity and protective efficacy of two mucosal/systemic vaccine regimens and investigated their effects on the rectal microbiome. Rhesus macaques were primed twice mucosally with replication-competent adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus (SIV) recombinants and boosted twice intramuscularly with ALVAC-SIV recombinant plus SIV gp120 protein or with DNA for SIV genes and rhesus interleukin-12 plus SIV gp120 protein. Controls received empty Ad5hr vector and alum adjuvant only. Both regimens elicited strong, comparable mucosal and systemic cellular and humoral immunity. Prevaccination rectal microbiomes of males and females differed and significantly changed over the course of immunization, most strongly in females after Ad5hr immunizations. Following repeated low-dose intrarectal SIV challenges, both vaccine groups exhibited modestly but significantly reduced acute viremia. Male and female controls exhibited similar acute viral loads; however, vaccinated females, but not males, exhibited lower levels of acute viremia, compared to same-sex controls. Few differences in adaptive immune responses were observed between the sexes. Striking differences in correlations of the rectal microbiome of males and females with acute viremia and immune responses associated with protection were seen and point to effects of the microbiome on vaccine-induced immunity and viremia control. Our study clearly demonstrates direct effects of a mucosal SIV vaccine regimen on the rectal microbiome and validates our previously reported SIV vaccine-induced sex bias. Sex and the microbiome are critical factors that should not be overlooked in vaccine design and evaluation.IMPORTANCE Differences in HIV pathogenesis between males and females, including immunity postinfection, have been well documented, as have steroid hormone effects on the microbiome, which is known to influence mucosal immune responses. Few studies have applied this knowledge to vaccine trials. We investigated two SIV vaccine regimens combining mucosal priming immunizations and systemic protein boosting. We again report a vaccine-induced sex bias, with female rhesus macaques but not males displaying significantly reduced acute viremia. The vaccine regimens, especially the mucosal primes, significantly altered the rectal microbiome. The greatest effects were in females. Striking differences between female and male macaques in correlations of prevalent rectal bacteria with viral loads and potentially protective immune responses were observed. Effects of the microbiome on vaccine-induced immunity and viremia control require further study by microbiome transfer. However, the findings presented highlight the critical importance of considering effects of sex and the microbiome in vaccine design and evaluation.

Analysis of Complement-Mediated Lysis of Simian Immunodeficiency Virus (SIV) and SIV-Infected Cells Reveals Sex Differences in Vaccine-Induced Immune Responses in Rhesus Macaques.

An effective human immunodeficiency virus (HIV) vaccine has yet to be developed, and defining immune correlates of protection against HIV infection is of paramount importance to inform future vaccine design. The complement system is a component of innate immunity that can directly lyse pathogens and shape adaptive immunity. To determine if complement lysis of simian immunodeficiency virus (SIV) and/or SIV-infected cells represents a protective immune correlate against SIV infection, sera from previously vaccinated and challenged rhesus macaques were analyzed for the induction of antibody-dependent complement-mediated lysis (ADCML). Importantly, the vaccine regimen, consisting of a replication-competent adenovirus type 5 host-range mutant SIV recombinant prime followed by a monomeric gp120 or oligomeric gp140 boost, resulted in overall delayed SIV acquisition only in females. Here, sera from all vaccinated animals induced ADCML of SIV and SIV-infected cells efficiently, regardless of sex. A modest correlation of SIV lysis with a reduced infection rate in males but not females, together with a reduced peak viremia in all animals boosted with gp140, suggested a potential for influencing protective efficacy. Gag-specific IgG and gp120-specific IgG and IgM correlated with SIV lysis in females, while Env-specific IgM correlated with SIV-infected cell lysis in males, indicating sex differences in vaccine-induced antibody characteristics and function. In fact, gp120/gp140-specific antibody functional correlates between antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and ADCML as well as the gp120-specific IgG glycan profiles and the corresponding ADCML correlations varied depending on the sex of the vaccinees. Overall, these data suggest that sex influences vaccine-induced antibody function, which should be considered in the design of globally effective HIV vaccines in the future.IMPORTANCE An HIV vaccine would thwart the spread of HIV infection and save millions of lives. Unfortunately, the immune responses conferring universal protection from HIV infection are poorly defined. The innate immune system, including the complement system, is an evolutionarily conserved, basic means of protection from infection. Complement can prevent infection by directly lysing incoming pathogens. We found that vaccination against SIV in rhesus macaques induces antibodies that are capable of directing complement lysis of SIV and SIV-infected cells in both sexes. We also found sex differences in vaccine-induced antibody species and their functions. Overall, our data suggest that sex affects vaccine-induced antibody characteristics and function and that males and females might require different immune responses to protect against HIV infection. This information could be used to generate highly effective HIV vaccines for both sexes in the future.

Influence of Plasma Cell Niche Factors on the Recruitment and Maintenance of IRF4hi Plasma Cells and Plasmablasts in Vaccinated, Simian Immunodeficiency Virus-Infected Rhesus Macaques with Low and High Viremia.

In a recent study, we found that protection following simian immunodeficiency virus (SIV) exposure correlated with rectal plasma cell frequency in vaccinated female rhesus macaques. We sought to determine if the same macaques maintained high mucosal plasma cell frequencies postinfection and if this translated to reduced viremia. Although delayed SIV acquisition did not predict subsequent viral control, alterations existed in the distribution of plasma cells and plasmablasts between macaques that exhibited high or low viremia. Flow cytometric analysis of cells from rectal biopsy specimens, bone marrow, and mesenteric lymph nodes of vaccinated infected, unvaccinated infected, and uninfected macaques identified two main IRF4hi subsets of interest: CD138+ plasma cells, and CD138- plasmablasts. In rectal tissue, plasma cell frequency positively correlated with plasma viremia and unvaccinated macaques had increased plasma cells and plasmablasts compared to vaccinated animals. Likewise, plasmablast frequency in the mesenteric lymph node correlated with viremia. However, in bone marrow, plasmablast frequency negatively correlated with viremia. Accordingly, low-viremic macaques had a higher frequency of both bone marrow IRF4hi subsets than did animals with high viremia. Significant reciprocal relationships between rectal and bone marrow plasmablasts suggested that efficient trafficking to the bone marrow as opposed to the rectal mucosa was linked to viral control. mRNA expression analysis of proteins involved in establishment of plasma cell niches in sorted bone marrow and rectal cell populations further supported this model and revealed differential mRNA expression patterns in these tissues. IMPORTANCE: As key antibody producers, plasma cells and plasmablasts are critical components of vaccine-induced immunity to human immunodeficiency virus type 1 (HIV-1) in humans and SIV in the macaque model; however, few have attempted to examine the role of these cells in viral suppression postinfection. Our results suggest that plasmablast trafficking to and retention in the bone marrow play a previously unappreciated role in viral control and contrast the potential contribution of mucosal plasma cells to mediate protection at sites of infection with that of bone marrow plasmablasts and plasma cells to control viremia during chronic infection. Manipulation of niche factors influencing the distribution and maintenance of these critical antibody-secreting cells may serve as potential therapeutic targets to enhance antiviral responses postvaccination and postinfection.