Adjuvanted Protein Vaccine Research Articles

An intranasal nanoparticle vaccine elicits protective immunity against Mycobacterium tuberculosis

Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3+ KLRG1− lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4+ T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4+ T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille–Calmette–Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis.

Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q)and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting invivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.

Intranasal nanoparticle vaccine elicits protective immunity against Mycobacterium tuberculosis

Abstract Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in the development stage. One of the primary challenges associated with mucosal vaccines has been the identification and characterization of safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. Intranasal administration of this vaccine provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille–Calmette–Guérin (BCG) upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain. Protection against the bacterium is likely due to CXCR3+ lung resident T cells that are known to be important for the control of bacterial infection. Intranasal delivery also induces IFNγ secreting CD4+ T cells in lung which is necessary for intracellular bactericidal activity. Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that provides durable protection from infection by M. tuberculosis.

Therapeutic efficacy against Mycobacterium tuberculosis using ID93 and liposomal adjuvant formulations.

Mycobacterium tuberculosis (M.tb) has led to approximately 1.3 million deaths globally in 2020 according to the World Health Organization (WHO). More effective treatments are therefore required to prevent the transmission of M.tb. Although Bacille Calmette–Guérin (BCG), a prophylactic vaccine against M.tb, already exists, other vaccines are being developed that could help boost BCG’s noted incomplete protection. This includes ID93 + GLA-SE, an adjuvanted protein vaccine which is being tested in Phase 2 clinical trials. The aim of this study was to test new lipid-based adjuvant formulations with ID93 in the context of a therapeutic vaccine, which we hypothesize would act as an adjunct to drug treatment and provide better outcomes, such as survival, than drug treatment alone. The recent success of another adjuvanted recombinant protein vaccine, M72 + AS01E (GlaxoSmithKline Biologicals), which after 3 years provided approximately 50% efficacy against TB pulmonary disease, is paving the way for new and potentially more effective vaccines. We show that based on selected criteria, including survival, T helper 1 cytokine responses, and resident memory T cells in the lung, that a liposomal formulation of GLA with QS-21 (GLA-LSQ) combined with ID93 provided enhanced protection over drug treatment alone.

#56 Does Cytomegalovirus (CMV) Status Impact the Host Response to SARS-CoV-2?

BackgroundCytomegalovirus (CMV) seropositivity has a significant immunomodulating over the life course, both with respect to responses to infections and responses to vaccines. For example, influenza vaccine response is known from multivariate analyses to be reduced by CMV seropositivity in elderly individuals. In patients with COVID-19, evidence suggests there are interactions between SARS-CoV-2 and CMV. CMV seropositives may have more severe COVID disease, and patients recovering from COVID often reactivate latent CMV, leading to end-organ CMV disease. This study aimed to test two hypotheses: 1) that vectored vaccines based on attenuated arenaviruses demonstrate equivalent immunogenicity to adjuvanted protein subunit spike (S) vaccines in a guinea pig vaccine model; 2) that CMV seropositivity impacts the immune response to S protein-based vaccines.MethodGuinea pigs (n=3 per group) were vaccinated with a 2-dose series of an attenuated arenavirus (Pichinde virus) S protein-vectored vaccine by intranasal (IN; n=4) or intramuscular (IM) route at a dose of 1x10^5 PFU. A 2-dose series of Addavax-adjuvanted S protein (RBD) vaccine (20 μg) was administered in parallel by IM or subcutaneous (SC) route. Matched control animals (Pichinde vector-only, or adjuvant only) were sham-vaccinated. The Pichinde-vector virus can be engineered with certain points of interest. It then can be transfected into other cells in the body. Blood was collected pre-vaccination and post-vaccination for CMV serology, and for SARS-CoV-2 S protein-specific ELISA, and for western blot analyses. Vaccine comparisons were conducted in CMV seronegative and CMV seropositive animals, to assess for an impact of CMV seropositivity on COVID vaccine response.ResultsNine of 25 animals tested positive for CMV antibodies prior to vaccination; 6 were in the S protein vaccine group, allowing comparison of the impact of CMV seropositivity to immune response compared to CMV negatives. COVID S ELISA antibodies were comparably induced by both vaccination routes. The magnitude of the Pichinde vectored vaccine response was similar to adjuvanted recombinant protein vaccine. When CMV-seropositive animals receiving any S protein vaccine (n=6) were compared to CMV-seronegatives (n=7), there was a trend toward decreased reciprocal RBD ELISA titer (p=0.17, Mann-Whitney). However, we noted that IN vaccination was associated with a strikingly lower ELISA titer (640) in a subset of CMV-seropositives.ConclusionIN administration of a Pichinde-vectored S vaccine demonstrated equivalent immunogenicity to an adjuvanted protein vaccine in guinea pigs, justifying the continued development of this novel mucosal vaccine strategy. However, pre-existing CMV seropositivity was associated with a trend toward a reduced response. CMV positivity may impact the immune response to S protein subunit vaccines delivered by IN route. Future studies in the guinea pig will focus on transplacental antibody transfer following COVID immunization of pregnant dams. Future clinical studies should examine the role of CMV serostatus both in COVID vaccine response and in disease pathogenesis.

Early Post-Vaccination Gene Signatures Correlate With the Magnitude and Function of Vaccine-Induced HIV Envelope-Specific Plasma Antibodies in Infant Rhesus Macaques.

A better understanding of the impact of early innate immune responses after vaccine priming on vaccine-elicited adaptive immune responses could inform rational design for effective HIV vaccines. The current study compared the whole blood molecular immune signatures of a 3M-052-SE adjuvanted HIV Env protein vaccine to a regimen combining the adjuvanted Env protein with simultaneous administration of a modified Vaccinia Ankara vector expressing HIV Env in infant rhesus macaques at days 0, 1, and 3 post vaccine prime. Both vaccines induced a rapid innate response, evident by elevated inflammatory plasma cytokines and altered gene expression. We identified 25 differentially-expressed genes (DEG) on day 1 compared to day 0 in the HIV protein vaccine group. In contrast, in the group that received both the Env protein and the MVA-Env vaccine only two DEG were identified, implying that the MVA-Env modified the innate response to the adjuvanted protein vaccine. By day 3, only three DEG maintained altered expression, indicative of the transient nature of the innate response. The DEG represented immune pathways associated with complement activation, type I interferon and interleukin signaling, pathogen sensing, and induction of adaptive immunity. DEG expression on day 1 was correlated to Env-specific antibody responses, in particular antibody-dependent cytotoxicity responses at week 34, and Env-specific follicular T helper cells. Results from network analysis supported the interaction of DEG and their proteins in B cell activation. These results emphasize that vaccine-induced HIV-specific antibody responses can be optimized through the modulation of the innate response to the vaccine prime.

Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates

The development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we show that mRNA-LNP immunization compared to protein immunization elicits either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope or HIV-1 Env gp160 induces durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 μg are immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.

Flavivirus DNA Vaccine Design and Adjuvant Selection.

A DNA vaccine is a plasmid encoding a vaccine antigen together with an efficient eukaryotic promoter to drive protein expression. The chief problem of DNA vaccines has been their suboptimal immunogenicity in humans. Many different flaviviruses infect and cause serious illness and even death in humans, but human vaccines are not available against most of the relevant flaviviruses with the exception of Japanese encephalitis virus. DNA vaccines are easy and fast to produce at relatively low cost, do not require handling of dangerous pathogens, are stable at room temperature allowing for low-cost storage and transportation, and are highly versatile, allowing for rapid changes in coding sequence design and synthesis. This makes a DNA vaccine approach ideally suited for development as a broad-based flavivirus vaccine platform. However, to be useful as a flavivirus prophylactic vaccine platform in humans, a method would need to be found to enhance DNA vaccine immunogenicity without the need for the cumbersome and expensive equipment involved with electroporation. We describe here a protocol used to test different adjuvants with flavivirus DNA vaccines to determine an optimal formulation. An optimal regimen involving a DNA adjuvanted vaccine prime followed by an adjuvanted protein vaccine boost is described and can be applied by readers to solve barriers to the development of other DNA vaccines where immunogenicity is a problem.

Recombinant subunit vaccines protect guinea pigs from lethal Ebola virus challenge

Ebola virus (EBOV) is among the deadliest pathogens known to man causing infrequent outbreaks of hemorrhagic disease. In humans, the case fatality rates in the outbreaks can reach 90%. During the West African epidemic almost 30,000 people were infected and of these over 11,000 fatalities were reported. Currently, we are facing an uncontained larger outbreak in the Democratic Republic of the Congo. Even though EBOV was discovered in 1976, extensive efforts to develop countermeasures, particularly therapeutics and vaccines, started late and there is still no FDA-approved product available. Nevertheless, one candidate vaccine, the rVSV-ZEBOV, is being used in clinical trials during the current outbreak with the hope of ending the human transmission chains. However, adverse reactions to administration of some EBOV vaccines have been reported; therefore, we have developed a safe and efficacious formulation of insect-cell derived adjuvanted protein vaccines. Vaccine candidates containing the EBOV glycoprotein with or without matrix proteins VP24 and VP40 formulated with one of three different adjuvants were tested in guinea pigs for immunogenicity and efficacy against lethal EBOV challenge. The results demonstrated that these vaccine candidates engendered high titers of antigen-specific antibodies in immunized animals and two of these vaccine candidates afforded complete or nearly complete protection against lethal challenge. Interestingly, we found a sex bias in partially protected immunized groups with male guinea pigs succumbing to disease and females surviving. In summary, we developed a safe and immunogenic adjuvanted subunit vaccine uniformly protective against EBOV disease in guinea pigs.

Baseline immune profile by CyTOF can predict response to an investigational adjuvanted vaccine in elderly adults

BackgroundMass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry.MethodsIn this study, we applied CyTOF to comprehensively characterize the circulating immune cell populations in elderly individuals both before and after administration of an investigational adjuvanted protein vaccine against respiratory syncytial virus (RSV) in a Phase 1a trial. Antigen-specific T cell responses to RSV by IFNγ ELISPOT had been observed in most but not all recipients in the highest dose cohort in this trial. Here, CyTOF was used to characterize the cellular response profile of ELISPOT responders and non-responders in this vaccine dose cohort.ResultsBoth CD4+ and CD8+ T cell antigen-specific IFNγ responses were observed. Principal components analysis revealed baseline differences between responders and non-responders, including differences in activated (HLA-DR+) CD4+ and CD8+ T cells, which were higher in non-responders versus responders. Using viSNE to analyze RSV-responsive CD4+ and CD8+ T cells, we also found increased expression of HLA-DR, CCR7, CD127 and CD69 in non-responders versus responders.ConclusionsHigh parameter CyTOF can help profile immune components associated with differential vaccine responsiveness.

Development of a thermostable nanoemulsion adjuvanted vaccine against tuberculosis using a design-of-experiments approach

BackgroundAdjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE).Materials and methodsIn this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations.ResultsWe identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies.ConclusionThese efforts will aid in the development of a platform formulation for use with other similar vaccines.

Inflammatory monocytes regulate Th1 oriented immunity to CpG adjuvanted protein vaccines through production of IL-12

Due to their capacity to skew T cell responses towards Th1 oriented immunity, oligonucleotides containing unmethylated CpG motifs (CpG) have emerged as interesting adjuvants for vaccination. Whereas the signalling pathways in response to CpG mediated TLR9 activation have been extensively documented at the level of the individual cell, little is however known on the precise identity of the innate immune cells that govern T cell priming and polarisation to CpG adjuvanted protein antigens in vivo. In this study, we demonstrate that optimal induction of Th1 oriented immunity to CpG adjuvanted protein vaccines requires the coordinated actions of conventional DCs and of monocytes. Whilst conventional DCs were required for antigen presentation and initial T cell priming, monocytes constitute the main source of the Th1 polarising cytokine IL-12.

Advanced Kinetic Analysis as a Tool for Formulation Development and Prediction of Vaccine Stability

We have used a protein‐based vaccine, a live virus vaccine, and an experimental adjuvant to evaluate the utility of an advanced kinetic modeling approach for stability prediction. The modeling approach uses a systematic and simple procedure for the selection of the most appropriate kinetic equation to describe the degradation rate of compounds subjected to accelerated conditions. One‐step and two‐step reactions with unlimited combinations of kinetic models were screened for the three products under evaluation. The most appropriate mathematical model for a given product was chosen based on the values of residual sum of squares and the weight parameter w. A relatively simple n‐th order kinetic model best fitted the degradation of an adjuvanted protein vaccine with a prediction error lower than 10%. A more complex two‐step model was required to describe inactivation of a live virus vaccine under normal and elevated storage temperatures. Finally, an autocatalytic‐type kinetic model best fitted the degradation of an oil‐in‐water adjuvant formulation. The modeling approach described here could be used for vaccine stability prediction, expiry date estimation, and formulation selection. To the best of our knowledge, this is the first report describing a global kinetic analysis of degradation of vaccine components with high prediction accuracy.

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1

BackgroundMalaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans.MethodsFluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated.ResultsAnalyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity.ConclusionsThe results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

Protective Th1 immune responses against chronic toxoplasmosis induced by a protein–protein vaccine combination but not by its DNA–protein counterpart

Vaccine-induced protection against toxoplasmosis is correlated with cellular immune responses to Toxoplasma gondii, both in animals and man. The goal of the current study was to evaluate whether the combination of a recombinant protein and a plasmid DNA vaccine could offer an advantage over the protein mixture, and protect outbred mice against infection with T. gondii. To this purpose, the chimeric protein rEC2, encoding antigenic fragments of surface-associated proteins MIC2, MIC3 and SAG1, was combined with pGRA7 plasmid DNA or rGRA7 protein. High levels of antibodies were elicited by both vaccine formulations. The protein–DNA vaccine elicited a polarized Th1/Th2 immune response, characterized by IFN-γ and IL-10, and afforded low protection (24%) against brain cyst formation. In contrast, the protein–protein vaccine elicited a Th1-focused immune response, characterized by IFN-γ and IL-2 production, conferring a strong protection (79%) against brain cyst formation in chronic toxoplasmosis. We show here that GERBU adjuvanted protein vaccines confer better protection against toxoplasmosis than the protein–DNA heterologous vaccine.

Alteration of epitope recognition pattern in Ag85B and ESAT‐6 has a profound influence on vaccine‐induced protection against Mycobacterium tuberculosis

To analyze the effect of vaccine delivery systems on antigen recognition and vaccine efficacy, we compared immune responses in mice immunized either with an adenovirus vector expressing a fusion of Ag85B and ESAT-6 or with the recombinant fusion protein in a liposomal adjuvant. Both vaccines induced high levels of antigen-specific IFN-gamma production. The adjuvanted protein vaccine induced primarily a CD4 T cell response directed to the epitope Ag85B(241-255) and gave efficient protection against subsequent Mycobacterium tuberculosis infection. In contrast, the adenoviral construct induced a strong CD8 response predominantly targeted to the epitope ESAT-6(15-29) and no significant protection against infection. Vaccination with the protein vaccine resulted in highly accelerated recall of Ag85B(241-255)-specific T cells immediately post M. tuberculosis challenge whereas the ESAT-6(15-29) epitope was barely recognized during infection. Delivery of the viral construct in cationic liposomes switched the immune response to a protective one dominated by CD4 T cells targeted to the Ag85B(241-255) epitope. These data demonstrate that the nature of the T cell response to a vaccine antigen is more important than its magnitude with respect to protective efficacy and that vaccine-mediated changes in immunodominance can result in T cell responses of limited relevance during the natural infection.

Rationale and perspectives on the success of vaccination against bovine herpesvirus-1

Several characteristics of BHV-1 have contributed to the successful development of both conventional and marker vaccines. BHV-1 is a stable virus, which grows to high titers in vitro, has a limited host range and causes acute viremic infections. Furthermore, the protective antigens, as well as the antigens that are suitable as marker, are present in the predominant virus isolates and induce significant and long-lasting immune responses, both in naïve and in previously vaccinated animals. In many parts of the world including North-America control of BHV-1 is achieved by vaccination with conventional attenuated or inactivated vaccines. With parts of Europe being BHV-1 free, the ability to differentiate infected from vaccinated animals has become critical as a trading tool. Live and killed gE-deleted marker vaccines are now widely used in Europe, in combination with gE-based diagnostic tests to monitor cattle. However, several issues remain to be resolved. BHV-1 causes latency, which creates a need for stringent management practices in case eradication is to be achieved. Since intramuscular delivery with a syringe and needle leads to considerable tissue damage, needle-free delivery methods should be adopted for beef cattle. Furthermore, conventional inactivated and attenuated vaccines are less efficacious in neonates, so alternative vaccine types such as CpG adjuvanted protein vaccines or DNA vaccines are required for effective vaccination of this age group.