Characterization of Flu MOSAIC nanoparticle vaccine candidate using high performance size-exclusion chromatography to support vaccine process development.

This report describes an application of analytical high performance size exclusion chromatography with UV and Fluorescent detection (HPSEC-UV/FLR) method that enabled a bridging from research vaccine candidate discovery (His-tagged model) to clinical product development (Non-His-tagged molecules). HPSEC measurement can accurately determine the total trimer-to-pentamer molar ratio by either titration evaluation during the nanoparticle being assembled or dissociation during a well-formed nanoparticle being dis-assembled. Through experimental design with small sample consumptions, HPSEC can provide a quick determination on the nanoparticle assembling efficiency which can therefore guide the buffer optimization for an assembly, from His-tagged model nanoparticle, to non-His-tagged clinical development product.

Development and validation of a mass spectrometry based analytical method to quantify the ratios in hemagglutinin trimers in quadrivalent influenza nanoparticle vaccine - FluMos-v1.

A quadrivalent influenza nanoparticle vaccine (FluMos-v1) offers long-lasting protection against multiple influenza virus strains and is composed of four strains of hemagglutinin trimer (HAT) assembled around a pentamer core. Here we report an LC-MS/MS analytical development and validation method to measure the percentage of each HAT component in FluMos-v1.

Analytical characterization of broadly neutralizing antibody CAP256LS heavy chain clipping during manufacturing development.

Broadly neutralizing antibody (bNAb) CAP256-VRC26.25 (abbreviated CAP256LS), a human IgGI monoclonal antibody targeting the V1V2 site of the HIV-1 envelope, has demonstrated high therapeutic potential as a broadly neutralizing monoclonal antibody against HIV-1. During the process development, a heavy chain fragmentation (clipping) was observed, that led to a relative potency reduction.

Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed.

Antigen expression determines adenoviral vaccine potency independent of IFN and STING signaling.

Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag).

Type I interferon-dependent activation of NK cells by rAd28 or rAd35, but not rAd5, leads to loss of vector-insert expression.

Vaccines constructed from rare-serotype recombinant adenovirus vectors (rAd) such as rAd serotype 28 (rAd28) and rAd35 are currently being explored as alternatives to rAd5-based vaccines because they circumvent the problems with pre-existing immunity that complicate the effectiveness of rAd5 vaccines. However, previous work has demonstrated that the immunogenicity of rAd28 and rAd35 is substantially lower than rAd5. Here we show that rAd28 and rAd35 increase apoptosis of antigen presenting cells (APCs), such as monocytes, relative to rAd5 and mock infected controls.

Comparative analysis of the magnitude, quality, phenotype, and protective capacity of simian immunodeficiency virus gag-specific CD8+ T cells following human-, simian-, and chimpanzee-derived recombinant adenoviral vector immunization.

Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8(+) T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. In this study we show low seroreactivity in humans against simian- (sAd11, sAd16) or chimpanzee-derived (chAd3, chAd63) compared with human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype, and protective capacity of CD8(+) T cell responses in mice vaccinated with rAds encoding SIV Gag.

Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.

The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies.

Gene-based vaccination with a mismatched envelope protects against simian immunodeficiency virus infection in nonhuman primates.

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally.

Type I IFN induced by adenovirus serotypes 28 and 35 has multiple effects on T cell immunogenicity.

Recombinant adenovirus (rAd) vectors are being investigated as vaccine delivery vehicles in preclinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. In this study, we compared the infectivity and cell stimulatory capacity of recombinant adenovirus serotype 5 (rAd5), recombinant adenovirus serotype 28 (rAd28), and recombinant adenovirus serotype 35 (rAd35) in association with their respective immunogenicity profiles.

Decreased pre-existing Ad5 capsid and Ad35 neutralizing antibodies increase HIV-1 infection risk in the Step trial independent of vaccination.

Background: The Step trial raised the possibility that uncircumcised men with pre-existing Ad5 neutralizing antibodies carried an increased risk of HIV infection after vaccination. Thus, understanding Ad seropositivity in humans is important to the development of an AIDS vaccine. Here, we analyze the impact of different Ad5-specific neutralizing antibodies on immune function and clinical outcome.

Adenovirus type-35 vectors block human CD4+ T-cell activation via CD46 ligation.

Recombinant adenoviruses (rAds) based on types 5 (rAd5) and 35 (rAd35) have emerged as important vaccine delivery vectors in clinical testing for a variety of pathogens. A major difference between these vectors is their binding to cellular receptors used for infection. Whereas rAd5 binds coxsackie-adenovirus receptor (CAR), rAd35 binds the complement regulatory protein CD46.

Characterization of human adenovirus 35 and derivation of complex vectors.

Background: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery.

Results: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined.

CD8+ T cell responses following replication-defective adenovirus serotype 5 immunization are dependent on CD11c+ dendritic cells but show redundancy in their requirement of TLR and nucleotide-binding oligomerization domain-like receptor signaling.

Replication-defective adenovirus serotype 5 (rAd5) is the most potent recombinant vector for eliciting CD8 T cell responses in humans. In this study, the innate mechanisms that influence T cell responses following rAd5 immunization were assessed in mice. Using rAd5 expressing enhanced GFP (eGFP-rAd5), we show that rAd5 transfects CD11c(+) dendritic cells (DCs) in draining lymph nodes in vivo following s.

Potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28.

Adenovirus vaccine vectors derived from rare human serotypes have been shown to be less potent than serotype 5 (Ad5) at inducing immune responses to encoded antigens. To identify highly immunogenic adenovirus vectors, we assessed pro-inflammatory cytokine expression, binding to the CD46 receptor, and immunogenicity. Species D adenoviruses uniquely suppressed pro-inflammatory cytokines and induced high levels of type I interferon.

Differential specificity and immunogenicity of adenovirus type 5 neutralizing antibodies elicited by natural infection or immunization.

A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination.

Delivery of human immunodeficiency virus vaccine vectors to the intestine induces enhanced mucosal cellular immunity.

Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4(+) T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity.

Replication-defective adenovirus vectors with multiple deletions do not induce measurable vector-specific T cells in human trials.

The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4(+) T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured.

Enhanced induction of intestinal cellular immunity by oral priming with enteric adenovirus 41 vectors.

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by the rapid onset of intestinal T-cell depletion that initiates the progression to AIDS. The induction of protective immunity in the intestinal mucosa therefore represents a potentially desirable feature of a preventive AIDS vaccine. In this study, we have evaluated the ability of an enteric adenovirus, recombinant adenovirus 41 (rAd41), to elicit intestinal and systemic immune responses by different immunization routes, alone or in combination with rAd5.

Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes.

Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels.

Mechanism of ad5 vaccine immunity and toxicity: fiber shaft targeting of dendritic cells.

Recombinant adenoviral (rAd) vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5) vector administration in a human gene therapy trial.

Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41.

Replication-defective adenovirus vectors, primarily developed from serotype 5 (Ad5) viruses, have been widely used for gene transfer and vaccination approaches. Vectors based on other serotypes of adenovirus could be used in conjunction with, or in place of, Ad5 vectors. In this study, Ad41, an enteric adenovirus usually described as 'non-cultivable' or 'fastidious,' has been successfully cloned, rescued and propagated on 293-ORF6 cells.

Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector.

Background: The development of an effective human immunodeficiency virus (HIV) vaccine is a high global priority. Here, we report the safety, tolerability, and immunogenicity of a replication-defective recombinant adenovirus serotype 5 (rAd5) vector HIV-1 candidate vaccine.

Methods: The vaccine is a mixture of 4 rAd5 vectors that express HIV-1 subtype B Gag-Pol fusion protein and envelope (Env) from subtypes A, B, and C.

Subgroup B and F fiber chimeras eliminate normal adenovirus type 5 vector transduction in vitro and in vivo.

Altering adenovirus vector (Ad vector) targeting is an important goal for a variety of gene therapy applications and involves eliminating or reducing the normal tropism of a vector and retargeting through a distinct receptor-ligand pathway. The first step of Ad vector infection is high-affinity binding to a target cellular receptor. For the majority of adenoviruses and Ad vectors, the fiber capsid protein serves this purpose, binding to the coxsackievirus and adenovirus receptor (CAR) present on a variety of cell types.