The Vaccinia Virus (VACV) B1 and Cellular VRK2 Kinases Promote VACV Replication Factory Formation through Phosphorylation-Dependent Inhibition of VACV B12.

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia virus protein B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1, unlike other DNA viruses. Employing loss-of-function analysis, we demonstrate that vaccinia virus's dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining coprecipitation of B12 by B1 and VRK2. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analyses of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-dependent manner.IMPORTANCE Constraints placed on viral genome size require that these pathogens must employ sophisticated, yet parsimonious mechanisms to effectively integrate with host cell signaling pathways. Poxviruses are no exception and employ several methods to balance these goals, including encoding single proteins that impact multiple downstream pathways. This study focuses on the vaccinia virus B1 protein kinase, an enzyme that promotes virus replication at multiple phases of the viral lifecycle. Herein, we demonstrate that in addition to its previously characterized functions, B1 inhibits vaccinia virus B12 protein via a phosphorylation-dependent mechanism and that this function of B1 can be complemented by the cellular B1 homolog VRK2. Combined with previous data implicating functional overlap between B1 and an additional cellular B1 homolog, VRK1, these data provide evidence of how poxviruses can be multifaceted in their mimicry of cellular proteins through the consolidation of functions of both VRK1 and VRK2 within the viral B1 protein kinase.

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.

Swine influenza A virus: challenges and novel vaccine strategies.

Swine Influenza A Virus (IAV-S) imposes a significant impact on the pork industry and has been deemed a significant threat to global public health due to its zoonotic potential. The most effective method of preventing IAV-S is vaccination. While there are tremendous efforts to control and prevent IAV-S in vulnerable swine populations, there are considerable challenges in developing a broadly protective vaccine against IAV-S. These challenges include the consistent diversification of IAV-S, increasing the strength and breadth of adaptive immune responses elicited by vaccination, interfering maternal antibody responses, and the induction of vaccine-associated enhanced respiratory disease after vaccination. Current vaccination strategies are often not updated frequently enough to address the continuously evolving nature of IAV-S, fail to induce broadly cross-reactive responses, are susceptible to interference, may enhance respiratory disease, and can be expensive to produce. Here, we review the challenges and current status of universal IAV-S vaccine research. We also detail the current standard of licensed vaccines and their limitations in the field. Finally, we review recently described novel vaccines and vaccine platforms that may improve upon current methods of IAV-S control.

Immunogenicity and Protective Efficacy of Dose-Sparing Epigraph Vaccine against H3 Swine Influenza A Virus.

Swine influenza A virus (IAV-S) is a highly prevalent and transmissible pathogen infecting worldwide swine populations. Our previous work has shown that the computationally derived vaccine platform, Epigraph, can induce broadly cross-reactive and durable immunity against H3 IAV-S in mice and swine. Therefore, in this study, we assess the immunogenicity and protective efficacy of the Epigraph vaccine at increasingly lower doses to determine the minimum dose required to maintain protective immunity against three genetically divergent H3 IAV-S. We assessed both antibody and T cell responses and then challenged with three H3N2 IAV-S derived from either Cluster IV(A), Cluster I, or the 2010.1 "human-like" cluster and assessed protection through reduced pathology, reduced viral load in the lungs, and reduced viral shedding from nasal swabs. Overall, we observed a dose-dependent effect where the highest dose of Epigraph protected against all three challenges, the middle dose of Epigraph protected against more genetically similar IAV-S, and the lowest dose of Epigraph only protected against genetically similar IAV-S. The results of these studies can be used to continue developing a broadly protective and low-dose vaccine against H3 IAV-S.

Multivalent Epigraph Hemagglutinin Vaccine Protects against Influenza B Virus in Mice.

Influenza B virus is a respiratory pathogen that contributes to seasonal epidemics, accounts for approximately 25% of global influenza infections, and can induce severe disease in young children. While vaccination is the most commonly used method of preventing influenza infections, current vaccines only induce strain-specific responses and have suboptimal efficacy when mismatched from circulating strains. Further, two influenza B virus lineages have been described, B/Yamagata-like and B/Victoria-like, and the limited cross-reactivity between the two lineages provides an additional barrier in developing a universal influenza B virus vaccine. Here, we report a novel multivalent vaccine using computationally designed Epigraph hemagglutinin proteins targeting both the B/Yamagata-like and B/Victoria-like lineages. When compared to the quadrivalent commercial vaccine, the Epigraph vaccine demonstrated increased breadth of neutralizing antibody and T cell responses. After lethal heterologous influenza B virus challenge, mice immunized with the Epigraph vaccine were completely protected against both weight loss and mortality. The superior cross-reactive immunity conferred by the Epigraph vaccine immunogens supports their continued investigation as a universal influenza B virus vaccine.

Lipid nanoparticle-encapsulated DNA vaccine confers protection against swine and human-origin H1N1 influenza viruses.

In 2009, a novel swine-origin H1N1 virus emerged, causing a pandemic. The virus, known as H1N1pdm09, quickly displaced the circulating H1 lineage and became the dominant seasonal influenza A virus subtype infecting humans. Human-to-swine spillovers of the H1N1pdm09 have occurred frequently, and each occurrence has led to sustained transmission of the human-origin H1N1pdm09 within swine populations. In the present study, we developed a lipid nanoparticle-based DNA vaccine (LNP-DNA) containing the hemagglutinin gene of a swine-origin H1N1pdm09. In pigs, this LNP-DNA vaccine induced a robust antibody response after a single intramuscular immunization and protected the pigs against challenge infection with the homologous swine-origin H1N1pdm09 virus. In a mouse model, the LNP-DNA vaccine induced antibody and T-cell responses and protected mice against lethal challenge with a mouse-adapted human-origin H1N1pdm09 virus. These findings demonstrate the potential of the LNP-DNA vaccine to protect against both swine- and human-origin H1N1pdm09 viruses. IMPORTANCE: Swine influenza A virus (IAV) is widespread and causes significant economic losses to the swine industry. Moreover, bidirectional transmission of IAV between swine and humans commonly occurs. Once introduced into the swine population, human-origin IAV often reassorts with endemic swine IAV, resulting in reassortant viruses. Thus, it is imperative to develop a vaccine that is not only effective against IAV strains endemic in swine but also capable of preventing the spillover of human-origin IAV. In this study, we developed a lipid nanoparticle-encapsulated DNA plasmid vaccine (LNP-DNA) that demonstrates efficacy against both swine- and human-origin H1N1 viruses. The LNP-DNA vaccines are non-infectious and non-viable, meeting the criteria to serve as a vaccine platform for rapidly updating vaccines. Collectively, this LNP-DNA vaccine approach holds great potential for alleviating the impact of IAV on the swine industry and preventing the emergence of reassortant IAV strains.

Heme oxygenase-1 regulates the immune response to influenza virus infection and vaccination in aged mice.

Underlying mechanisms of individual variation in severity of influenza infection and response to vaccination are poorly understood. We investigated the effect of reduced heme oxygenase-1 (HO-1) expression on vaccine response and outcome of influenza infection. HO-1-deficient and wild-type (WT) mice (kingdom, Animalia; phylum, Chordata; genus/species, Mus musculus) were infected with influenza virus A/PR/8/34 with or without prior vaccination with an adenoviral-based influenza vaccine. A genome-wide association study evaluated the expression of single-nucleotide polymorphisms (SNPs) in the HO-1 gene and the response to influenza vaccination in healthy humans. HO-1-deficient mice had decreased survival after influenza infection compared to WT mice (median survival 5.5 vs. 6.5 d, P=0.016). HO-1-deficient mice had impaired production of antibody following influenza vaccination compared to WT mice (mean antibody titer 869 vs. 1698, P=0.02). One SNP in HO-1 and one SNP in the constitutively expressed isoform HO-2 were independently associated with decreased antibody production after influenza vaccination in healthy human volunteers (P=0.017 and 0.014, respectively). HO-1 deficient mice were paired with sex- and age-matched WT controls. HO-1 affects the immune response to both influenza infection and vaccination, suggesting that therapeutic induction of HO-1 expression may represent a novel adjuvant to enhance influenza vaccine effectiveness.

Existing Evidence for Influenza B Virus Adaptations to Drive Replication in Humans as the Primary Host.

Influenza B virus (IBV) is one of the two major types of influenza viruses that circulate each year. Unlike influenza A viruses, IBV does not harbor pandemic potential due to its lack of historical circulation in non-human hosts. Many studies and reviews have highlighted important factors for host determination of influenza A viruses. However, much less is known about the factors driving IBV replication in humans. We hypothesize that similar factors influence the host restriction of IBV. Here, we compile and review the current understanding of host factors crucial for the various stages of the IBV viral replication cycle. While we discovered the research in this area of IBV is limited, we review known host factors that may indicate possible host restriction of IBV to humans. These factors include the IBV hemagglutinin (HA) protein, host nuclear factors, and viral immune evasion proteins. Our review frames the current understanding of IBV adaptations to replication in humans. However, this review is limited by the amount of research previously completed on IBV host determinants and would benefit from additional future research in this area.

Adenoviral-vectored epigraph vaccine elicits robust, durable, and protective immunity against H3 influenza A virus in swine.

Current methods of vaccination against swine Influenza A Virus (IAV-S) in pigs are infrequently updated, induce strain-specific responses, and have a limited duration of protection. Here, we characterize the onset and duration of adaptive immune responses after vaccination with an adenoviral-vectored Epigraph vaccine. In this longitudinal study we observed robust and durable antibody responses that remained above protective titers six months after vaccination. We further identified stable levels of antigen-specific T cell responses that remained detectable in the absence of antigen stimulation. Antibody isotyping revealed robust class switching from IgM to IgG induced by Epigraph vaccination, while the commercial comparator vaccine failed to induce strong antibody class switching. Swine were challenged six months after initial vaccination, and Epigraph-vaccinated animals demonstrated significant protection from microscopic lesion development in the trachea and lungs, reduced duration of viral shedding, lower presence of infectious virus and viral antigens in the lungs, and significant recall of antigen-specific T cell responses following challenge. The results obtained from this study are useful in determining the kinetics of adaptive immune responses after vaccination with adjuvanted whole inactivated virus vaccines compared to adenoviral vectored vaccines and contribute to the continued efforts of creating a universal IAV-S vaccine.

Generation of a Kupffer cell-evading adenovirus for systemic and liver-directed gene transfer.

As much as 90% of an intravenously (i.v.) injected dose of adenovirus serotype 5 (Ad5) is absorbed and destroyed by liver Kupffer cells. Viruses that escape these cells can then transduce hepatocytes after binding factor X (FX). Given that interactions with FX and Kupffer cells are thought to occur on the Ad5 hexon protein, we replaced its exposed hypervariable regions (HVR) with those from Ad6. When tested in vivo in BALB/c mice and in hamsters, the Ad5/6 chimera mediated >10 times higher transduction in the liver. This effect was not due to changes in FX binding. Rather, Ad5/6 appeared to escape Kupffer cell uptake as evidenced by producing no Kupffer cell death in vivo, not requiring predosing in vivo, and being phagocytosed less efficiently by macrophages in vitro compared to Ad5. When tested as a helper-dependent adenovirus (Ad) vector, Ad5/6 mediated higher luciferase and factor IX transgene expression than either helper-dependent adenoviral 5 (HD-Ad5) or HD-Ad6 vectors. These data suggest that the Ad5/6 hexon-chimera evades Kupffer cells and may have utility for systemic and liver-directed therapies.

Adenoviral-Vectored Centralized Consensus Hemagglutinin Vaccine Provides Broad Protection against H2 Influenza a Virus.

Several influenza pandemics have occurred in the past century, one of which emerged in 1957 from a zoonotic transmission of H2N2 from an avian reservoir into humans. This pandemic caused 2-4 million deaths and circulated until 1968. Since the disappearance of H2N2 from human populations, there has been waning immunity against H2, and this subtype is not currently incorporated into seasonal vaccines. However, H2 influenza remains a pandemic threat due to consistent circulation in avian reservoirs. Here, we describe a method of pandemic preparedness by creating an adenoviral-vectored centralized consensus vaccine design against human H2 influenza. We also assessed the utility of serotype-switching to enhance the protective immune responses seen with homologous prime-boosting strategies. Immunization with an H2 centralized consensus showed a wide breadth of antibody responses after vaccination, protection against challenge with a divergent human H2 strain, and significantly reduced viral load in the lungs after challenge. Further, serotype switching between two species C adenoviruses enhanced protective antibody titers after heterologous boosting. These data support the notion that an adenoviral-vectored H2 centralized consensus vaccine has the ability to provide broadly cross-reactive immune responses to protect against divergent strains of H2 influenza and prepare for a possible pandemic.

Expanding Mouse-Adapted Yamagata-like Influenza B Viruses in Eggs Enhances In Vivo Lethality in BALB/c Mice.

Despite the yearly global impact of influenza B viruses (IBVs), limited host range has been a hurdle to developing a readily accessible small animal disease model for vaccine studies. Mouse-adapting IBV can produce highly pathogenic viruses through serial lung passaging in mice. Previous studies have highlighted amino acid changes throughout the viral genome correlating with increased pathogenicity, but no consensus mutations have been determined. We aimed to show that growth system can play a role in mouse-adapted IBV lethality. Two Yamagata-lineage IBVs were serially passaged 10 times in mouse lungs before expansion in embryonated eggs or Madin-Darby canine kidney cells (London line) for use in challenge studies. We observed that virus grown in embryonated eggs was significantly more lethal in mice than the same virus grown in cell culture. Ten additional serial lung passages of one strain again showed virus grown in eggs was more lethal than virus grown in cells. Additionally, no mutations in the surface glycoprotein amino acid sequences correlated to differences in lethality. Our results suggest growth system can influence lethality of mouse-adapted IBVs after serial lung passaging. Further research can highlight improved mechanisms for developing animal disease models for IBV vaccine research.

An epitope-optimized human H3N2 influenza vaccine induces broadly protective immunity in mice and ferrets.

There is a crucial need for an improved H3N2 influenza virus vaccine due to low vaccine efficacy rates and increased morbidity and mortality associated with H3N2-dominated influenza seasons. Here, we utilize a computational design strategy to produce epitope-optimized, broadly cross-reactive H3 hemagglutinins in order to create a universal H3N2 influenza vaccine. The Epigraph immunogens are designed to maximize the viral population frequency of epitopes incorporated into the immunogen. We compared our Epigraph H3 vaccine to the traditional egg-based inactivated influenza vaccine from 2018-19, FluZone. Epigraph vaccination-induced stronger cross-reactive antibody responses than FluZone against 18 H3N2 viruses isolated from 1968 to 2019 in both mice and ferrets, with protective hemagglutination inhibition titers against 93-100% of the contemporary H3N2 strains compared to only 27% protection measured from FluZone. In addition, Epigraph vaccination-induced strong cross-reactive T-cell immunity which significantly contributes to protection against lethal influenza virus infection. Finally, Epigraph vaccination protected ferrets from influenza disease after challenge with two H3N2 viruses. The superior cross-reactive immunity induced by these Epigraph immunogens supports their development as a universal H3N2 influenza vaccine.

Strategies Targeting Hemagglutinin as a Universal Influenza Vaccine.

Influenza virus has significant viral diversity, both through antigenic drift and shift, which makes development of a vaccine challenging. Current influenza vaccines are updated yearly to include strains predicted to circulate in the upcoming influenza season, however this can lead to a mismatch which reduces vaccine efficacy. Several strategies targeting the most abundant and immunogenic surface protein of influenza, the hemagglutinin (HA) protein, have been explored. These strategies include stalk-directed, consensus-based, and computationally derived HA immunogens. In this review, we explore vaccine strategies which utilize novel antigen design of the HA protein to improve cross-reactive immunity for development of a universal influenza vaccine.

Epigraph hemagglutinin vaccine induces broad cross-reactive immunity against swine H3 influenza virus.

Influenza A virus infection in swine impacts the agricultural industry in addition to its zoonotic potential. Here, we utilize epigraph, a computational algorithm, to design a universal swine H3 influenza vaccine. The epigraph hemagglutinin proteins are delivered using an Adenovirus type 5 vector and are compared to a wild type hemagglutinin and the commercial inactivated vaccine, FluSure. In mice, epigraph vaccination leads to significant cross-reactive antibody and T-cell responses against a diverse panel of swH3 isolates. Epigraph vaccination also reduces weight loss and lung viral titers in mice after challenge with three divergent swH3 viruses. Vaccination studies in swine, the target species for this vaccine, show stronger levels of cross-reactive antibodies and T-cell responses after immunization with the epigraph vaccine compared to the wild type and FluSure vaccines. In both murine and swine models, epigraph vaccination shows superior cross-reactive immunity that should be further investigated as a universal swH3 vaccine.

Species D Adenoviruses as Oncolytic Viral Vectors.

Oncolytic adenoviruses (Ad) have shown promising results in the therapeutic treatment of cancer. Ad type 5 (Ad5) is the most extensively utilized Ad type. However, several limitations exist to using Ad5 as an oncolytic virus, including high levels of anti-Ad5 neutralizing antibodies in the population, binding of the Ad5 hexon to blood coagulation factor X leading to liver sequestration and toxicity, and reduced expression of the primary receptor CAR on many tumors. Here, we use in vitro methods to explore the oncolytic potential of four alternative Ad types (Ad26, 28, 45, and 48) belonging to the species D Ad subgroup and developed replication-competent species D Ads expressing the human sodium iodide symporter protein (hNIS) for combination radiovirotherapy. We evaluated the species D Ad vectors transduction, replication, cytotoxicity, and gene expression in six different cancer cell lines. Species D Ads showed the greatest transduction and cytotoxic killing in the SKBR3 breast cancer cells, followed by 293, A549, and HepG2 cells, however the cytotoxicity was less than the wild type Ad5 virus. In contrast, species D Ads showed limited transduction and cytotoxicity in the Hela and SKOV3 cancer cell lines. These species D Ad vectors also successfully expressed the hNIS gene during infection leading to increased iodide uptake in multiple cancer cell lines. These results, the low seroprevalence of anti-species D antibodies, and the lack of binding to blood coagulation FX, support further exploration of species D Ads as alternative oncolytic adenoviruses against multiple types of cancer.

A Decade in Review: A Systematic Review of Universal Influenza Vaccines in Clinical Trials during the 2010 Decade.

On average, there are 3-5 million severe cases of influenza virus infections globally each year. Seasonal influenza vaccines provide limited protection against divergent influenza strains. Therefore, the development of a universal influenza vaccine is a top priority for the NIH. Here, we report a comprehensive summary of all universal influenza vaccines that were tested in clinical trials during the 2010-2019 decade. Of the 1597 studies found, 69 eligible clinical trials, which investigated 27 vaccines, were included in this review. Information from each trial was compiled for vaccine target, vaccine platform, adjuvant inclusion, clinical trial phase, and results. As we look forward, there are currently three vaccines in phase III clinical trials which could provide significant improvement over seasonal influenza vaccines. This systematic review of universal influenza vaccine clinical trials during the 2010-2019 decade provides an update on the progress towards an improved influenza vaccine.

Characterization of a Species E Adenovirus Vector as a Zika virus vaccine.

The development of a safe and efficacious Zika virus (ZIKV) vaccine remains a global health priority. In our previous work, we developed an Adenovirus vectored ZIKV vaccine using a low-seroprevalent human Adenovirus type 4 (Ad4-prM-E) and compared it to an Ad5 vector (Ad5-prM-E). We found that vaccination with Ad4-prM-E leads to the development of a strong anti-ZIKV T-cell response without eliciting significant anti-ZIKV antibodies, while vaccination with Ad5-prM-E leads to the development of both anti-ZIKV antibody and T-cell responses in C57BL/6 mice. However, both vectors conferred protection against ZIKV infection in a lethal challenge model. Here we continued to characterize the T-cell biased immune response observed in Ad4 immunized mice. Vaccination of BALB/c mice resulted in immune correlates similar to C57BL/6 mice, confirming that this response is not mouse strain-specific. Vaccination with an Ad4 expressing an influenza hemagglutinin (HA) protein resulted in anti-HA T-cell responses without the development of significant anti-HA antibodies, indicating this unique response is specific to the Ad4 serotype rather than the transgene expressed. Co-administration of a UV inactivated Ad4 vector with the Ad5-prM-E vaccine led to a significant reduction in anti-ZIKV antibody development suggesting that this serotype-specific immune profile is capsid-dependent. These results highlight the serotype-specific immune profiles elicited by different Adenovirus vector types and emphasize the importance of continued characterization of these alternative Ad serotypes.

Modification of adenoviral vectors with polyethylene glycol modulates in vivo tissue tropism and gene expression.

Polyethylene glycol (PEG) is a hydrophilic polymer that has been used to coat adenoviral (Ad) vectors to improve their pharmacology. To analyze the effects of PEG on Ad5 tropism, Ad5 was covalently modified with different sizes of PEG and in vitro and in vivo transduction was analyzed. All tested PEGs ablated in vitro transduction. When protein C (PC) and factors VII, IX, and X were added, only factors IX and X increased transduction by the PEGylated vectors with the largest effect by X. Inactivation of these factors with warfarin drastically reduced liver transduction in mice by the PEGylated vectors after intravenous (i.v.) injection. Ad5 conjugated with 5 kd PEG maintained normal liver transduction while conjugation with larger 20 and 35 kd PEGs significantly reduced liver transduction. When intraperitoneal (i.p.) injection was tested, Ad transduced the peritoneum efficiently with only low level liver transduction. When Ad5 was modified with 5 kd PEG, peritoneal transduction was reduced and the virus preferentially transduced the liver. These data demonstrate the effects of different sizes of PEG on in vivo Ad tropism and suggest that this approach may be useful in retargeting and detargeting Ad in vivo.

Dose Effects of Recombinant Adenovirus Immunization in Rodents.

Recombinant adenovirus type 5 (rAd) has been used as a vaccine platform against many infectious diseases and has been shown to be an effective vaccine vector. The dose of the vaccine varies significantly from study to study, making it very difficult to compare immune responses and vaccine efficacy. This study determined the immune correlates induced by serial dilutions of rAd vaccines delivered intramuscularly (IM) and intranasally (IN) to mice and rats. When immunized IM, mice had substantially higher antibody responses at the higher vaccine doses, whereas, the IN immunized mice showed a lower response to the higher rAd vaccine doses. Rats did not show dose-dependent antibody responses to increasing vaccine doses. The IM immunized mice and rats also showed significant dose-dependent T cell responses to the rAd vaccine. However, the T cell immunity plateaued in both mice and rats at 109 and 1010 vp/animal, respectively. Additionally, the highest dose of vaccine in mice and rats did not improve the T cell responses. A final vaccine analysis using a lethal influenza virus challenge showed that despite the differences in the immune responses observed in the mice, the mice had very similar patterns of protection. This indicates that rAd vaccines induced dose-dependent immune responses, especially in IM immunized animals, and that immune correlates are not as predictive of protection as initially thought.