Preclinical Immunogenicity Research Articles

Preclinical evaluation of a candidate naked plasmid DNA vaccine against SARS-CoV-2

New generation plasmid DNA vaccines may be a safe, fast and simple emergency vaccine platform for preparedness against emerging viral pathogens. Applying platform optimization strategies, we tested the pre-clinical immunogenicity and protective effect of a candidate DNA plasmid vaccine specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The DNA vaccine induced spike-specific binding IgG and neutralizing antibodies in mice, rabbits, and rhesus macaques together with robust Th1 dominant cellular responses in small animals. Intradermal and intramuscular needle-free administration of the DNA vaccine yielded comparable immune responses. In a vaccination-challenge study of rhesus macaques, the vaccine demonstrated protection from viral replication in the lungs following intranasal and intratracheal inoculation with SARS-CoV-2. In conclusion, the candidate plasmid DNA vaccine encoding the SARS-CoV-2 spike protein is immunogenic in different models and confers protection against lung infection in nonhuman primates. Further evaluation of this DNA vaccine candidate in clinical trials is warranted.

Safety and Immunogenicity Analysis of a Newcastle Disease Virus (NDV-HXP-S) Expressing the Spike Protein of SARS-CoV-2 in Sprague Dawley Rats.

Despite global vaccination efforts, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and spread globally. Relatively high vaccination rates have been achieved in most regions of the United States and several countries worldwide. However, access to vaccines in low- and mid-income countries (LMICs) is still suboptimal. Second generation vaccines that are universally affordable and induce systemic and mucosal immunity are needed. Here we performed an extended safety and immunogenicity analysis of a second-generation SARS-CoV-2 vaccine consisting of a live Newcastle disease virus vector expressing a pre-fusion stabilized version of the spike protein (NDV-HXP-S) administered intranasally (IN), intramuscularly (IM), or IN followed by IM in Sprague Dawley rats. Local reactogenicity, systemic toxicity, and post-mortem histopathology were assessed after the vaccine administration, with no indication of severe local or systemic reactions. Immunogenicity studies showed that the three vaccination regimens tested elicited high antibody titers against the wild type SARS-CoV-2 spike protein and the NDV vector. Moreover, high antibody titers were induced against the spike of B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants of concern (VOCs). Importantly, robust levels of serum antibodies with neutralizing activity against the authentic SARS-CoV-2 USA‐WA1/2020 isolate were detected after the boost. Overall, our study expands the pre-clinical safety and immunogenicity characterization of NDV-HXP-S and reinforces previous findings in other animal models about its high immunogenicity. Clinical testing of this vaccination approach is ongoing in different countries including Thailand, Vietnam, Brazil and Mexico.

Development of an Inactivated Vaccine against SARS CoV-2.

The rapid spread of SARS-CoV-2 with its mutating strains has posed a global threat to safety during this COVID-19 pandemic. Thus far, there are 123 candidate vaccines in human clinical trials and more than 190 candidates in preclinical development worldwide as per the WHO on 1 October 2021. The various types of vaccines that are currently approved for emergency use include viral vectors (e.g., adenovirus, University of Oxford/AstraZeneca, Gamaleya Sputnik V, and Johnson & Johnson), mRNA (Moderna and Pfizer-BioNTech), and whole inactivated (Sinovac Biotech and Sinopharm) vaccines. Amidst the emerging cases and shortages of vaccines for global distribution, it is vital to develop a vaccine candidate that recapitulates the severe and fatal progression of COVID-19 and further helps to cope with the current outbreak. Hence, we present the preclinical immunogenicity, protective efficacy, and safety evaluation of a whole-virion inactivated SARS-CoV-2 vaccine candidate (ERUCoV-VAC) formulated in aluminium hydroxide, in three animal models, BALB/c mice, transgenic mice (K18-hACE2), and ferrets. The hCoV-19/Turkey/ERAGEM-001/2020 strain was used for the safety evaluation of ERUCoV-VAC. It was found that ERUCoV-VAC was highly immunogenic and elicited a strong immune response in BALB/c mice. The protective efficacy of the vaccine in K18-hACE2 showed that ERUCoV-VAC induced complete protection of the mice from a lethal SARS-CoV-2 challenge. Similar viral clearance rates with the safety evaluation of the vaccine in upper respiratory tracts were also positively appreciable in the ferret models. ERUCoV-VAC has been authorized by the Turkish Medicines and Medical Devices Agency and has now entered phase 3 clinical development (NCT04942405). The name of ERUCoV-VAC has been changed to TURKOVAC in the phase 3 clinical trial.

Immunogenicity of Botulinum Toxin Formulations: Potential Therapeutic Implications.

Botulinum neurotoxins (BoNTs) are proteins produced by bacteria of the Clostridium family. Upon oral ingestion, BoNT causes the neuroparalytic syndrome botulism. There are seven serotypes of BoNT (serotypes A-G); BoNT-A and BoNT-B are the botulinum toxin serotypes utilized for therapeutic applications. Treatment with BoNT injections is used to manage chronic medical conditions across multiple indications. As with other biologic drugs, immunogenicity after long-term treatment with BoNT formulations may occur, and repeated use can elicit antibody formation leading to clinical nonresponsiveness. Thus, approaching BoNT treatment of chronic conditions with therapeutic formulations that minimize stimulating the host immune response while balancing patient responsiveness to therapy is ideal. Immunogenicity is a clinical limitation in many settings that use biologic drugs for treatment, and clinically relevant immunogenicity reduction has been achieved through engineering smaller protein constructs and reducing unnecessary formulation components. A similar approach has influenced the evolution of BoNT formulations. Three BoNT-A products and one BoNT-B product have been approved by the Food and Drug Administration (FDA) for therapeutic use: onabotulinumtoxinA, abobotulinumtoxinA, incobotulinumtoxinA, and rimabotulinumtoxinB; a fourth BoNT-A product, daxibotulinumtoxinA, is currently under regulatory review. Additionally, prabotulinumtoxinA is a BoNT-A product that has been approved for aesthetic indications but not therapeutic use. Here, we discuss the preclinical and clinical immunogenicity data that exist within the scientific literature and provide a perspective for considering immunogenicity as a key factor in choice of BoNT formulation.

Comparability study of monocyte derived dendritic cells, primary monocytes, and THP1 cells for innate immune responses

Immunogenicity is one major challenge to the successful development of biotherapeutics because it could adversely affect PK/PD, safety, and efficacy. Preclinical immunogenicity risk assessment strategies and assays have been developed and implemented to screen and optimize discovery molecules. Internalization by antigen presenting cells (APC) and innate immune activation are initial prerequisite steps in eliciting immune responses to biotherapeutics. Dendritic cells (DC)- and monocyte-based assays are employed to interrogate such risks, and their value has been well documented in the literature. However, these assays have limited throughput, exhibit higher variability, and entail lengthy and complex procedures as they are based on primary cells such as peripheral blood mononuclear cells (PBMC) from individual donors. Herein, we investigated THP1 cells as surrogate cells to study APC internalization and innate immune activation. Comparability studies showed that THP1 cells could resemble innate immune responses of monocyte-derived DC and primary CD14+ monocytes using a panel of therapeutic antibodies. In addition, an automated high throughput THP1 internalization assay was qualified to enable risk assessment at pre‑lead stages. The results demonstrated that THP1 cells can be utilized to assess immunogenicity risk in a high throughput manner.

Preclinical immunogenicity testing using anti-drug antibody analysis of GX-G3, Fc-fused recombinant human granulocyte colony-stimulating factor, in rat and monkey models

Human granulocyte colony-stimulating factor (G-CSF, this study used Fc-fused recombinant G-CSF; GX-G3) is an important glycoprotein that stimulates the proliferation of granulocytes and white blood cells. Thus, G-CSF treatment has been considered as a crucial regimen to accelerate recovery from chemotherapy-induced neutropenia in cancer patients suffering from non-myeloid malignancy or acute myeloid leukemia. Despite the therapeutic advantages of G-CSF treatment, an assessment of its immunogenicity must be performed to determine whether the production of anti-G-CSF antibodies causes immune-related disorders. We optimized and validated analytical tools by adopting validation parameters for immunogenicity assessment. Using these validated tools, we analyzed serum samples from rats and monkeys injected subcutaneously with GX-G3 (1, 3 or 10 mg/kg once a week for 4 weeks followed by a 4-week recovery period) to determine immunogenicity response and toxicokinetic parameters with serum concentration of GX-G3. Several rats and monkeys were determined to be positive for anti-GX-G3 antibodies. Moreover, the immunogenicity response of GX-G3 was lower in monkeys than in rats, which was relevant to show less inhibition of toxicokinetic profiles in monkeys, at least 1 mg/kg administrated group, compared to rats. These results suggested the establishment and validation for analyzing anti-GX-G3 antibodies and measurement of serum levels of GX-G3 and anti-GX-G3 antibodies, which was related with toxicokinetic profiles. Taken together, this study provides immunogenicity assessment which is closely implicated with toxicokinetic study of GX-G3 in 4-week repeated administrated toxicological studies.

Managing the Impact of Immunogenicity in an Era of Immunotherapy: From Bench to Bedside.

Biotherapeutics have revolutionized our ability to treat life-threatening diseases. Despite clinical success, the use of biotherapeutics has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs). The multifactorial nature of immunogenicity has prevented a standardized approach for assessing this and each of the assessment methods developed so far does not exhibit high enough reliability to be used alone, due to limited predictiveness. This prompted the Roche Pharma Research and Early Development (pRED) Immunogenicity Working Group to establish an internal preclinical immunogenicity toolbox of in vitro/in vivo approaches and accompanying guidelines for a harmonized assessment and management of immunogenicity in early development. In this article, the complex factors influencing immunogenicity and their associated clinical ramifications are discussed to highlight the importance of an end-to-end approach conducted from lead optimization to clinical candidate selection. We then examine the impact of the resulting lead candidate categorization on the design and implementation of a multi-tiered ADA/immunogenicity assay strategy prior to phase I (entry into human) through early clinical development. Ultimately, the Immunogenicity Toolbox ensures that Roche pRED teams are equipped to address immunogenicity in a standardized manner, paving the way for lifesaving products with improved safety and efficacy.

Preclinical safety and immunogenicity of Streptococcus pyogenes (Strep A) peptide vaccines

We have developed two candidate vaccines to protect against multiple strains of Strep A infections. The candidates are combinatorial synthetic peptide vaccines composed of a M protein epitope (J8 or p*17) and a non-M protein epitope (K4S2). To enhance immunogenicity, each peptide is conjugated to the carrier protein CRM197 (CRM) and formulated with aluminium hydroxide adjuvant Alhydrogel (Alum) to make the final vaccines, J8-CRM + K4S2-CRM/Alum and p*17-CRM + K4S2-CRM/Alum. The safety and toxicity of each vaccine was assessed. Sprague Dawley rats were administered three intramuscular doses, over a six-week study with a 4-week recovery period. A control group received CRM only formulated with Alum (CRM/Alum). There was no evidence of systemic toxicity in the rats administered either vaccine. There was an associated increase in white blood cell, lymphocyte and monocyte counts, increased adrenal gland weights, adrenocortical hypertrophy, and increased severity of granulomatous inflammation at the sites of injection and the associated inguinal lymph nodes. These changes were considered non-adverse. All rats administered vaccine developed a robust and sustained immunological response. The absence of clinical toxicity and the development of an immunological response in the rats suggests that the vaccines are safe for use in a phase 1 clinical trial in healthy humans.

DEVELOPMENT AND PRE-CLINICAL STUDIES OF THE RECOMBINANT VACCINE "BETUVAX-COV-2" FOR PREVENTION OF COVID-19

A candidate vaccine containing a corpuscular adjuvant and a recombinant glycosylated RBD domain of the SARS- CoV-2 virus has successfully passed preclinical studies and is approved for clinical trials. In the course of preclinical studies, the safety and high immunogenicity of the developed drug was shown in experiments on animals, including primates.

Enabling Routine MHC-II-Associated Peptide Proteomics for Risk Assessment of Drug-Induced Immunogenicity.

Major histocompatibility complex-II (MHC-II)-Associated Peptide Proteomics (MAPPs) is a mass spectrometry-based approach to identify and relatively quantitate naturally processed and presented MHC-II-associated peptides that can potentially activate T cells and contribute to the immunogenicity of a drug. Acceptance of the MAPPs technology as an appropriate preclinical (and potentially clinical) immunogenicity risk assessment tool depends not only on its technical stability and robustness but also on the ability to compare results across experiments and donors. To this end, we developed a specialized MAPPs data processing pipeline, dataMAPPs, which presents complex mass spectrometric data sets in the form of heat maps (heatMAPPs), enabling rapid and convenient comparison between conditions and donors. A customized normalization procedure based on identified endogenous peptides standardizes signal intensities within and between donors and enables cross-experimental comparison. We evaluated the technical reproducibility of the MAPPs platform using tool compounds with respect to the most prominent experimental factors and found that the systematic biological differences across donors by far outweighed any technical source of variation. We illustrate the capability of the MAPPs platform to generate data that may be used for preclinical risk assessment of drug-induced immunogenicity and discuss its applicability in the clinics.

Specificity of the T Cell Response to Protein Biopharmaceuticals.

The anti-drug antibody (ADA) response is an undesired humoral response raised against protein biopharmaceuticals (BPs) which can dramatically disturb their therapeutic properties. One particularity of the ADA response resides in the nature of the immunogens, which are usually human(ized) proteins and are therefore expected to be tolerated. CD4 T cells initiate, maintain and regulate the ADA response and are therefore key players of this immune response. Over the last decade, advances have been made in characterizing the T cell responses developed by patients treated with BPs. Epitope specificity and phenotypes of BP-specific T cells have been reported and highlight the effector and regulatory roles of T cells in the ADA response. BP-specific T cell responses are assessed in healthy subjects to anticipate the immunogenicity of BP prior to their testing in clinical trials. Immunogenicity prediction, also called preclinical immunogenicity assessment, aims at identifying immunogenic BPs and immunogenic BP sequences before any BP injection in humans. All of the approaches that have been developed to date rely on the detection of BP-specific T cells in donors who have never been exposed to BPs. The number of BP-specific T cells circulating in the blood of these donors is therefore limited. T cell assays using cells collected from healthy donors might reveal the weak tolerance induced by BPs, whose endogenous form is expressed at a low level. These BPs have a complete human sequence, but the level of their endogenous form appears insufficient to promote the negative selection of autoreactive T cell clones. Multiple T cell epitopes have also been identified in therapeutic antibodies and some other BPs. The pattern of identified T cell epitopes differs across the antibodies, notwithstanding their humanized, human or chimeric nature. However, in all antibodies, the non-germline amino acid sequences mainly found in the CDRs appear to be the main driver of immunogenicity, provided they can be presented by HLA class II molecules. Considering the fact that the BP field is expanding to include new formats and gene and cell therapies, we face new challenges in understanding and mastering the immunogenicity of new biological products.

Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth.

A vaccine will likely be one of the key tools for ending the HIV-1/AIDS epidemic by preventing HIV-1 spread within uninfected populations and achieving a cure for people living with HIV-1. The currently prevailing view of the vaccine field is to introduce protective antibodies, nevertheless, a vaccine to be effective may need to harness protective T cells. We postulated that focusing a T-cell response on the most vulnerable regions of the HIV-1 proteome while maximizing a perfect match between the vaccine and circulating viruses will control HIV-1 replication. We currently use a combination of replication-deficient simian (chimpanzee) adenovirus and poxvirus modified vaccinia virus Ankara to deliver bivalent conserved-mosaic immunogens to human volunteers. Here, we exploit the mRNA platform by designing tetravalent immunogens designated as HIVconsvM, and demonstrate that mRNA formulated in lipid nanoparticles induces potent, broad and polyfunctional T-cell responses in a pre-clinical model. These results support optimization and further development of this vaccine strategy in experimental medicine trials in humans.

Biophysical characterization of polydisperse liposomal adjuvant formulations

Army Liposome Formulations (ALF) are potent adjuvants, of which there are two primary forms, lyophilized ALF (ALFlyo) containing monophosphoryl lipid A (MPLA) and ALF containing MPLA and QS21 (ALFQ). ALFlyo and ALFQ adjuvants are essential constituents of candidate vaccines for bacterial, viral, and parasitic diseases. They have been widely used in preclinical immunogenicity studies in small animals and non-human primates and are progressing to phase I/IIa clinical trials. ALFQ was prepared by adding saponin QS21 to small unilamellar liposome vesicles (SUVs) of ALF55 that contain 55 mol% cholesterol, whereas ALFlyo was created by reconstituting lyophilized SUVs of ALF43, consisting of 43 mol% cholesterol, in aqueous buffer solution. These formulations display heterogenous particle size distribution. Since biophysical characteristics of liposomes may impact their adjuvant potential, we characterized the particle size distribution and lamellarity of the individual liposome particles in ALFlyo and ALFQ formulations using cryo-electron microscopy and a newly developed MANTA technology. ALFlyo and ALFQ exhibited similar particle size distributions with liposomes ranging from 50 nm to several μm. However, fundamental differences were observed in the lamellar structures of the liposomes. ALFlyo displayed a greater number of multilamellar and multivesicular liposome particles, as compared to that in ALFQ, which was predominately unilamellar.

The Immune Response to the fVIII Gene Therapy in Preclinical Models.

Neutralizing antibodies to factor VIII (fVIII), referred to as “inhibitors,” remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product- and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liver-specific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pre-transplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.

A Systematic Approach to HIV-1 Vaccine Immunogen Selection.

A tremendous loss of financial and human resources from seven large-scale HIV vaccine efficacy trials suggest a need for a systematic approach to vaccine selection. We conducted a systematic analysis of three important envelope glycoprotein (Env) vaccine candidates: BG505 SOSIP.664, 1086.C gp140, and 1086.C gp120 to determine the most promising by comparing their structure and antigenicity. We found that the BG505 SOSIP trimer and 1086.C gp140 clearly outperformed the 1086.C gp120 monomer. BG505 SOSIP.664 bound the strongest to the most potent and broadest broadly neutralizing antibodies (bnAbs) PG9, PGT145, VRC01, and PGT121. Of interest, although BG505 SOSIP.664 did not bind to the CH58 mAb, 1086.C gp140 bound strongly to this mAb, which belongs to a class of non-neutralizing antibodies that may be protective based on correlates of protection studies of the RV144 HIV vaccine trial. The 1086.C gp120 monomer was the least antigenic of the three vaccine immunogens, binding the weakest to bnAbs and CH58 mAb. Taken together, the evidence provided here combined with previous preclinical immunogenicity and efficacy data strongly argue that the BG505 SOSIP.664 trimer and 1086.C gp140 are likely to be better vaccine immunogens than the monomeric 1086.C gp120, which was just recently tested and shown to be nonefficacious in a phase IIb/III trial. Thus, to best utilize our financial and valuable human resources, we propose a systematic approach by not only comparing structure and antigenicity, but also immunogenicity and efficacy of Env vaccine candidates in the preclinical phase to the selection of only the most promising vaccine candidates for clinical testing.

Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development

BackgroundCoronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. MethodsWe first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. FindingsHere we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. InterpretationMNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.

In vitro characterization and preclinical immunogenicity of Typhax, a typhoid fever protein capsular matrix vaccine candidate

ABSTRACT Typhax is an investigational typhoid fever vaccine candidate that was GMP manufactured applying Protein Capsular Matrix Vaccine (PCMV) technology. It consists of Vi polysaccharide antigen, derived from S. Typhi, non-covalently entrapped in a glutaraldehyde catalyzed cross-linked α-poly-L-lysine and CRM197 protein matrix. Analysis of Typhax determined the average molecular weight of the vaccine particles was approximately 6 x 106 Daltons, corresponding to particles containing 1–2 molecules of Vi polysaccharide and 10–20 molecules of CRM197 protein. The ratio of the concentration of Vi to CRM197 protein in Typhax is 2.4:1. Preclinical immunogenicity studies in mice demonstrated that Typhax was immunogenic and elicited a significant increase in anti-Vi IgG antibody titers following each immunization. The anti-Vi IgG antibody response elicited by Typhax in rabbits increased as the dose increased from 0.1 µg to 2.5 µg. Further, at the 2.5 and 10 µg dose levels, the anti-Vi IgG antibody titers increased after the second and third immunizations. At the 10 µg dose level, 100% of rabbits seroconverted. In the non-human primate (NHP) study, 100% seroconversion was observed at both 2.5 µg and 10 µg dose levels after the first immunization. A murine in vivo immunopotency study demonstrated that Typhax stored at 4°C was stable for at least 30 months. Collectively, the Typhax in vitro profile, preclinical immunogenicity studies, and rabbit toxicology study indicate that Typhax is a viable typhoid fever vaccine candidate for Phase 1 clinical trial evaluation.

Preclinical immunogenicity and safety of the cGMP-grade placental malaria vaccine PRIMVAC.

BackgroundVAR2CSA is the lead antigen for developing a vaccine that would protect pregnant women against placental malaria. A multi-system feasibility study has identified E. coli as a suitable bacterial expression platform allowing the production of recombinant VAR2CSA-DBL1x-2x (PRIMVAC) to envisage a prompt transition to current Good Manufacturing Practice (cGMP) vaccine production.MethodsExtensive process developments were undertaken to produce cGMP grade PRIMVAC to permit early phase clinical trials. PRIMVAC stability upon storage was assessed over up to 3 years. A broad toxicology investigation was carried out in rats allowing meanwhile the analysis of PRIMVAC immunogenicity.FindingsWe describe the successful cGMP production of 4. 65 g of PRIMVAC. PRIMVAC drug product was stable and potent for up to 3 years upon storage at −20 °C and showed an absence of toxicity in rats. PRIMVAC adjuvanted with Alhydrogel® or GLA-SE was able to generate antibodies able to recognize VAR2CSA expressed at the surface of erythrocytes infected with different strains. These antibodies also inhibit the interaction of the homologous NF54-CSA strain and to a lower extend of heterologous strains to CSA.InterpretationThis work paved the way for the clinical development of an easily scalable low cost effective vaccine that could protect against placental malaria and prevent an estimated 10,000 maternal and 200,000 infant deaths annually.FundThis work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF), Germany through Kreditanstalt für Wiederaufbau (KfW) (Reference No: 202060457) and through funding from Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Purified Inactivated Zika Vaccine Candidates Afford Protection against Lethal Challenge in Mice

In response to the 2016 global public health emergency of international concern announced by the World Health Organization surrounding Zika virus (ZIKV) outbreaks, we developed a purified inactivated Zika virus vaccine (PIZV) candidate from ZIKV strain PRVABC59, isolated during the outbreak in 2015. The virus isolate was plaque purified, creating six sub-isolated virus stocks, two of which were selected to generate PIZV candidates for preclinical immunogenicity and efficacy evaluation in mice. The alum-adjuvanted PIZV candidates were highly immunogenic in both CD-1 and AG129 mice after a 2-dose immunization. Further, AG129 mice receiving 2 doses of PIZV formulated with alum were fully protected against lethal ZIKV challenge and mouse immune sera elicited by the PIZV candidates were capable of neutralizing ZIKVs of both African and Asian genetic lineages in vitro. Additionally, passive immunization of naïve mice with ZIKV-immune serum showed strong positive correlation between neutralizing ZIKV antibody (NAb) titers and protection against lethal challenge. This study supported advancement of the PIZV candidate toward clinical development.

Preclinical immunogenicity and protective efficacy of an oral Helicobacter pylori inactivated whole cell vaccine and multiple mutant cholera toxin: A novel and non-toxic mucosal adjuvant

Mucosal vaccines against Helicobacter pylori consisting of either whole cell bacteria or recombinant antigens can induce immune protection against challenge in mice only when co-administrated with a strong mucosal adjuvant such as cholera toxin (CT) or Escherichia coli heat labile enterotoxin (LT). The strong enterotoxicity of these adjuvants however preclude their use in human vaccines. The recently developed multiple mutant CT (mmCT) is a strong, yet practically non-toxic novel mucosal adjuvant which here was admixed with a formalin-inactivated H. pylori whole cell vaccine (WCV) as a potential vaccine candidate against H. pylori infection. We report that intragastric immunizations with H. pylori WCV together with mmCT, similar to immunization with WCV together with CT, resulted in 50–125-fold reduction in colonization of H. pylori in the stomach of mice associated with rises in both serum IgG and intestinal-mucosal IgA anti-H. pylori antibody responses and strong T cell and IFNγ and IL-17A cytokine responses. Data presented in this study also supports that the proposed vaccine can be grown in a bioreactor and would be effective against infection caused by a multitude of pathogenic H. pylori strains isolated from patients from various continents. The results warrant immunization studies in humans to evaluate the safety, immunogenicity and efficacy of the proposed H. pylori WCV and mmCT.