Optimization of a Plasmodium falciparum circumsporozoite protein repeat vaccine using the tobacco mosaic virus platform.

Plasmodium falciparum vaccine RTS,S/AS01 is based on the major NPNA repeat and the C-terminal region of the circumsporozoite protein (CSP). RTS,S-induced NPNA-specific antibody titer and avidity have been associated with high-level protection in naïve subjects, but efficacy and longevity in target populations is relatively low. In an effort to improve upon RTS,S, a minimal repeat-only, epitope-focused, protective, malaria vaccine was designed. Repeat antigen copy number and flexibility was optimized using the tobacco mosaic virus (TMV) display platform. Comparing antigenicity of TMV displaying 3 to 20 copies of NPNA revealed that low copy number can reduce the abundance of low-affinity monoclonal antibody (mAb) epitopes while retaining high-affinity mAb epitopes. TMV presentation improved titer and avidity of repeat-specific Abs compared to a nearly full-length protein vaccine (FL-CSP). NPNAx5 antigen displayed as a loop on the TMV particle was found to be most optimal and its efficacy could be further augmented by combination with a human-use adjuvant ALFQ that contains immune-stimulators. These data were confirmed in rhesus macaques where a low dose of TMV-NPNAx5 elicited Abs that persisted at functional levels for up to 11 mo. We show here a complex association between NPNA copy number, flexibility, antigenicity, immunogenicity, and efficacy of CSP-based vaccines. We hypothesize that designing minimal epitope CSP vaccines could confer better and more durable protection against malaria. Preclinical data presented here supports the evaluation of TMV-NPNAx5/ALFQ in human trials.

Adjuvanted HIV-1 vaccine promotes antibody-dependent phagocytic responses and protects against heterologous SHIV challenge.

To augment HIV-1 pox-protein vaccine immunogenicity using a next generation adjuvant, a prime-boost strategy of recombinant modified vaccinia virus Ankara and multimeric Env gp145 was evaluated in macaques with either aluminum (alum) or a novel liposomal monophosphoryl lipid A (MPLA) formulation adsorbed to alum, ALFA. Binding antibody responses were robust and comparable between arms, while antibody-dependent neutrophil and monocyte phagocytotic responses were greatly enhanced by ALFA. Per-exposure vaccine efficacy against heterologous tier 2 SHIV mucosal challenge was 90% in ALFA-adjuvanted males (P = 0.002), while alum conferred no protection. Half of the ALFA-adjuvanted males remained uninfected after the full challenge series, which spanned seven months after the last vaccination. Antibody-dependent monocyte and neutrophil phagocytic responses both strongly correlated with protection. Significant sex differences in infection risk were observed, with much lower infection rates in females than males. In humans, MPLA-liposome-alum adjuvanted gp120 also increased HIV-1-specific phagocytic responses relative to alum. Thus, next-generation liposome-based adjuvants can drive vaccine elicited antibody effector activity towards potent phagocytic responses in both macaques and humans and these responses correlate with protection. Future protein vaccination strategies aiming to improve functional humoral responses may benefit from such adjuvants.

Design, Synthesis, and In Vivo Evaluation of C1-Linked 4,5-Epoxymorphinan Haptens for Heroin Vaccines.

In our continuing effort to develop effective anti-heroin vaccines as potential medications for the treatment of opioid use disorder, herein we present the design and synthesis of the haptens: 1-AmidoMorHap (1), 1-AmidoMorHap epimer (2), 1 Amido-DihydroMorHap (3), and 1 Amido-DihydroMorHap epimer (4). This is the first report of hydrolytically stable haptenic surrogates of heroin with the attachment site at the C1 position in the 4,5-epoxymorophinan nucleus. We prepared respective tetanus toxoid (TT)-hapten conjugates as heroin vaccine immunogens and evaluated their efficacy in vivo. We showed that all TT-hapten conjugates induced high antibody endpoint titers against the targets but only haptens 2 and 3 can induce protective effects against heroin in vivo. The epimeric analogues of these haptens, 1 and 4, failed to protect mice from the effects of heroin. We also showed that the in vivo efficacy is consistent with the results of the in vitro drug sequestration assay. Attachment of the linker at the C1 position induced antibodies with weak binding to the target drugs. Only TT-2 and TT-3 yielded antibodies that bound heroin and 6-acetyl morphine. None of the TT-hapten conjugates induced antibodies that cross-reacted with morphine, methadone, naloxone, or naltrexone, and only TT-3 interacted weakly with buprenorphine, and that subtle structural difference, especially at the C6 position, can vastly alter the specificity of the induced antibodies. This study is an important contribution in the field of vaccine development against small-molecule targets, providing proof that the chirality at C6 in these epoxymorphinans is a vital key to their effectiveness.

Impact of Th1 CD4 Follicular Helper T Cell Skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokine-dependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferon-induced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses.IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine.

Bivalent Conjugate Vaccine Induces Dual Immunogenic Response That Attenuates Heroin and Fentanyl Effects in Mice.

Opioid use disorders and fatal overdose due to consumption of fentanyl-laced heroin remain a major public health menace in the United States. Vaccination may serve as a promising potential remedy to combat accidental overdose and to mitigate the abuse potential of opioids. We previously reported the heroin and fentanyl monovalent vaccines carrying, respectively, a heroin hapten, 6-AmHap, and a fentanyl hapten, para-AmFenHap, conjugated to tetanus toxoid (TT). Herein, we describe the mixing of these antigens to formulate a bivalent vaccine adjuvanted with liposomes containing monophosphoryl lipid A (MPLA) adsorbed on aluminum hydroxide. Immunization of mice with the bivalent vaccine resulted in IgG titers of >105 against both haptens. The polyclonal sera bound heroin, 6-acetylmorphine, morphine, and fentanyl with dissociation constants (Kd) of 0.25 to 0.50 nM. Mice were protected from the anti-nociceptive effects of heroin, fentanyl, and heroin +9% (w/w) fentanyl. No cross-reactivity to methadone and buprenorphine was observed in vivo. Naloxone remained efficacious in immunized mice. These results highlighted the potential of combining TT-6-AmHap and TT-para-AmFenHap to yield an efficacious bivalent vaccine that could ablate heroin and fentanyl effects. This vaccine warrants further testing to establish its potential translatability to 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.

Novel Vaccine That Blunts Fentanyl Effects and Sequesters Ultrapotent Fentanyl Analogues.

Active immunization is an emerging potential modality to combat fatal overdose amid the opioid epidemic. In this study, we described the design, synthesis, formulation, and animal testing of an efficacious vaccine against fentanyl. The vaccine formulation is composed of a novel fentanyl hapten conjugated to tetanus toxoid (TT) and adjuvanted with liposomes containing monophosphoryl lipid A adsorbed on aluminum hydroxide. The linker and hapten N-phenyl-N-(1-(4-(3-(tritylthio)propanamido)phenethyl)piperidin-4-yl)propionamide were conjugated sequentially to TT using amine-N-hydroxysuccinimide-ester and thiol-maleimide reaction chemistries, respectively. Conjugation was facile, efficient, and reproducible with a protein recovery of >98% and a hapten density of 30-35 per carrier protein molecule. In mice, immunization induced high and robust antibody endpoint titers in the order of >106 against the hapten. The antisera bound fentanyl, carfentanil, cyclopropyl fentanyl, para-fluorofentanyl, and furanyl fentanyl in vitro with antibody-drug dissociation constants in the range of 0.36-4.66 nM. No cross-reactivity to naloxone, naltrexone, methadone, or buprenorphine was observed. In vivo, immunization shifted the antinociceptive dose-response curve of fentanyl to higher doses. Collectively, these preclinical results showcased the desired traits of a potential vaccine against fentanyl and demonstrated the feasibility of immunization to combat fentanyl-induced effects.

Impact of the expression system on the immune responses to self-assembling protein nanoparticles (SAPNs) displaying HIV-1 V1V2 loop.

The V1V2 loop of the Env protein is a major target for HIV-1 vaccine development because in multiple studies antibodies to this region correlated with protection. Although SAPNs expressed in E. coli elicited anti-V1V2 antibodies, the Env protein is heavily glycosylated. In this study the technology has been adapted for expression in mammalian cells. SAPNs containing a V1V2 loop from a B-subtype transmitter/founder virus were expressed in E. coli, ExpiCHO, and Expi293 cells. Independent of the expression host, particles were well-formed. All SAPNs raised high titers of V1V2-specific antibodies, however, SAPNE.coli induced a mainly anti-V1 response, while SAPNExpiCHO and SAPNExpi293 induced a predominantly anti-V2 response. In an ADCP assay, sera from animals immunized with the SAPNExpiCHO or SAPNExpi293 induced a significant increase in phagocytic activity. This novel way of producing SAPNs displaying glycosylated epitopes could increase the antibody titer, functional activity, and shift the immune response towards the desired pathway.

Comparison of immunogenicity and safety outcomes of a malaria vaccine FMP013/ALFQ in rhesus macaques (Macaca mulatta) of Indian and Chinese origin.

BACKGROUND: Indian-origin rhesus (InR) are preferred for research, but strict export restrictions continue to limit their use. Chinese-origin rhesus (ChR), although easier to procure, are genetically distinct from InR and differ in their immune response to infectious agents, such as the Simian Immunodeficiency Virus. The most advanced malaria vaccine, RTS,S (GlaxoSmithKline), is based on the circumsporozoite protein (CSP) of Plasmodium falciparum. The efficacy of RTS,S vaccine in the field remains low and short-lived; efforts are underway to improve CSP-based vaccines. Rhesus models can accelerate preclinical down-selection of the next generation of malaria vaccines. This study was used to determine if the safety and immunogenicity outcomes following vaccination with a CSP vaccine would differ in the InR and ChR models, given the genetic differences between the two sub-populations of rhesus. METHODS: The FMP013 vaccine, was composed of nearly full-length soluble P. falciparum CSP produced in Escherichia coli and was adjuvanted with the Army liposomal formulation (ALFQ). Three doses of the vaccine were administered in InR and ChR (n = 6) at 1-month intervals and the antibody and T cell responses were assessed. RESULTS: Local and systemic toxicity profile of FMP013 vaccine in InR and ChR were similar and they revealed that the FMP013 vaccine was safe and caused only mild and transient inflammatory adverse reactions. Following the first 2 vaccines, there was a slower acquisition of antibodies to the CSP repeat region in ChR. However after the 3rd vaccination the titers in the two models were comparable. The ChR group repeat-specific antibodies had higher avidity and ChR group showed higher inhibition of liver stage development activity compared to InR. There was no difference in T-cell responses to the FMP013 vaccine between the two models. CONCLUSIONS: A difference in the quality of serological responses was detected between the two sub-populations of rhesus. However, both models confirmed that FMP013/ALFQ vaccine was safe, highly immunogenic, elicited functional antibodies and T-cell responses. Overall, the data suggests that rhesus of Indian and Chinese origins can be interchangeably used to compare the safety and immunogenicity of next-generation of malaria vaccines and adjuvants.

Design and characterization of a self-assembling protein nanoparticle displaying HIV-1 Env V1V2 loop in a native-like trimeric conformation as vaccine antigen.

The RV144 HIV-1 clinical trial demonstrated modest vaccine efficacy and identified IgG antibodies against the Env V1V2 loop that inversely correlated with risk of infection. Based upon these results, we chose the Self-Assembling Protein Nanoparticle platform to present the V1V2 loop in a native-like conformation. We hypothesized this approach would lead to generation of conformation-specific IgG antibodies to V1V2. Our vaccine, V1V2-SHB-SAPN, was designed to present twenty copies of the V1V2 trimer. Particles were characterized for size, shape, and binding to monoclonal antibodies that recognize the V2 and V1V2 loops. Immunization induced IgG antibodies to V1, V2, V1V2 and to gp70V1V2 (AE/A244) capture antigens in mice. The presence of the Army Liposome Formulation induced a four-fold increase in IgG titers to gp70V1V2 and the adjuvanted V1V2-SHB-SAPN group had statistically higher IgG titers than sequence- and dose-matched V1V2 peptide controls. In conclusion, V1V2-SHB-SAPN vaccine presented the V1V2 loop in native-like conformation, as indicated by PGT145 binding, and induced high titers of IgG antibodies.

Saturated phospholipids are required for nano- to micron-size transformation of cholesterol-containing liposomes upon QS21 addition.

Liposomes containing cholesterol and monophosphoryl lipid A (such as ALFQ and AS01B) are vaccine adjuvants. During construction of the formulations, addition of QS21 to nano-size (50-100 nm) liposomes resulted in extremely large (up to ∼30 µm) liposomes in ALFQ, but AS01B liposomes remained small nano-vesicles. Here, we show that saturation of phospholipid chains is essential for production of large liposomes by QS21.

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate.

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.

Efficacy, but not antibody titer or affinity, of a heroin hapten conjugate vaccine correlates with increasing hapten densities on tetanus toxoid, but not on CRM197 carriers.

Vaccines against drugs of abuse have induced antibodies in animals that blocked the biological effects of the drug by sequestering the drug in the blood and preventing it from crossing the blood-brain barrier. Drugs of abuse are too small to induce antibodies and, therefore, require conjugation of drug hapten analogs to a carrier protein. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Previously, we have shown that 1 (MorHap), a heroin/morphine hapten, conjugated to tetanus toxoid (TT) and mixed with liposomes containing monophosphoryl lipid A [L(MPLA)] as adjuvant, partially blocked the antinociceptive effects of heroin in mice. Herein, we extended those findings, demonstrating greatly improved vaccine induced antinociceptive effects up to 3% mean maximal potential effect (%MPE). This was obtained by evaluating the effects of vaccine efficacy of hapten 1 vaccine conjugates with varying hapten densities using two different commonly used carrier proteins, TT and cross-reactive material 197 (CRM197). Immunization of mice with these conjugates mixed with L(MPLA) induced very high anti-1 IgG peak levels of 400-1500 µg/mL that bound to both heroin and its metabolites, 6-acetylmorphine and morphine. Except for the lowest hapten density for each carrier, the antibody titers and affinity were independent of hapten density. The TT carrier based vaccines induced long-lived inhibition of heroin-induced antinociception that correlated with increasing hapten density. The best formulation contained TT with the highest hapten density of ≥30 haptens/TT molecule and induced %MPE of approximately 3% after heroin challenge. In contrast, the best formulation using CRM197 was with intermediate 1 densities (10-15 haptens/CRM197 molecule), but the %MPE was approximately 13%. In addition, the chemical synthesis of 1, the optimization of the conjugation method, and the methods for the accurate quantification of hapten density are described.

Synthesis and immunological effects of heroin vaccines.

Three haptens have been synthesized with linkers for attachment to carrier macromolecules at either the piperidino-nitrogen or via an introduced 3-amino group. Two of the haptens, with a 2-oxopropyl functionality at either C6, or at both the C3 and C6 positions on the 4,5-epoxymorphinan framework, as well as the third hapten (DiAmHap) with diamido moieties at both the C3 and C6 positions, should be much more stable in solution, or in vivo in a vaccine, than a hapten with an ester in one of those positions, as found in many heroin-based haptens. A "classical" opioid synthetic scheme enabled the formation of a 3-amino-4,5-epoxymorphinan which could not be obtained using palladium chemistry. Our vaccines are aimed at the reduction of the abuse of heroin and, as well, at the reduction of the effects of its predominant metabolites, 6-acetylmorphine and morphine. One of the haptens, DiAmHap, has given interesting results in a heroin vaccine and is clearly more suited for the purpose than the other two haptens.

Restricted valency (NPNA)n repeats and junctional epitope-based circumsporozoite protein vaccines against Plasmodium falciparum.

The Circumsporozoite Protein (CSP) of Plasmodium falciparum contains an N-terminal region, a conserved Region I (RI), a junctional region, 25-42 copies of major (NPNA) and minor repeats followed by a C-terminal domain. The recently approved malaria vaccine, RTS,S/AS01 contains NPNAx19 and the C-terminal region of CSP. The efficacy of RTS,S against natural infection is low and short-lived, and mapping epitopes of inhibitory monoclonal antibodies may allow for rational improvement of CSP vaccines. Tobacco Mosaic Virus (TMV) was used here to display the junctional epitope (mAb CIS43), Region I (mAb 5D5), NPNAx5, and NPNAx20 epitope of CSP (mAbs 317 and 580). Protection studies in mice revealed that Region I did not elicit protective antibodies, and polyclonal antibodies against the junctional epitope showed equivalent protection to NPNAx5. Combining the junctional and NPNAx5 epitopes reduced immunogenicity and efficacy, and increasing the repeat valency to NPNAx20 did not improve upon NPNAx5. TMV was confirmed as a versatile vaccine platform for displaying small epitopes defined by neutralizing mAbs. We show that polyclonal antibodies against engineered VLPs can recapitulate the binding specificity of the mAbs and immune-focusing by reducing the structural complexity of an epitope may be superior to immune-broadening as a vaccine design approach. Most importantly the junctional and restricted valency NPNA epitopes can be the basis for developing highly effective second-generation malaria vaccine candidates.

First-in-human assessment of safety and immunogenicity of low and high doses of Plasmodium falciparum malaria protein 013 (FMP013) administered intramuscularly with ALFQ adjuvant in healthy malaria-naïve adults.

The global burden of malaria remains substantial. Circumsporozoite protein (CSP) has been demonstrated to be an effective target antigen, however, improvements that offer more efficacious and more durable protection are still needed. In support of research and development of next-generation malaria vaccines, Walter Reed Army Institute of Research (WRAIR) has developed a CSP-based antigen (FMP013) and a novel adjuvant ALFQ (Army Liposome Formulation containing QS-21). We present a single center, open-label, dose-escalation Phase 1 clinical trial to evaluate the safety and immunogenicity of the FMP013/ALFQ malaria vaccine candidate. In this first-in-human evaluation of both the antigen and adjuvant, we enrolled ten subjects; five received 20 µg FMP013 / 0.5 mL ALFQ (Low dose group), and five received 40 µg FMP013 / 1.0 mL ALFQ (High dose group) on study days 1, 29, and 57. Adverse events and immune responses were assessed during the study period. The clinical safety profile was acceptable and there were no serious adverse events. Both groups exhibited robust humoral and cellular immunological responses, and compared favorably with historical responses reported for RTS,S/AS01. Based on a lower reactogenicity profile, the 20 µg FMP013 / 0.5 mL ALFQ (Low dose) was selected for follow-on efficacy testing by controlled human malaria infection (CHMI) with a separate cohort. Trial Registration:Clinicaltrials.gov Identifier NCT04268420 (Registered February 13, 2020).

The diversity of HIV-1 fights against vaccine efficacy: how self-assembling protein nanoparticle technology may fight back.

An efficacious HIV-1 vaccine has remained an elusive target for almost 40 years. The sheer diversity of the virus is one of the major roadblocks for vaccine development. HIV-1 frequently mutates and various strains predominate in different geographic regions, making the development of a globally applicable vaccine extremely difficult. Multiple approaches have been taken to overcome the issue of viral diversity, including sequence optimization, development of consensus and mosaic sequences and the use of different prime-boost approaches. To develop an efficacious vaccine, these approaches may need to be combined. One way to potentially synergize these approaches is to use a rationally designed protein nanoparticle that allows for the native-like presentation of antigens, such as the self-assembling protein nanoparticle.

Synthesis and immunological effects of C14-linked 4,5-epoxymorphinan analogues as novel heroin vaccine haptens.

Active immunization is being explored as a potential therapeutic to combat accidental overdose and to mitigate the abuse potential of opioids. Hapten design is one of the crucial factors that determines the efficacy of a candidate vaccine to substance abuse and remains one of the most active areas of research in vaccine development. Herein we report for the first time the synthesis of three novel opiate surrogates with the linker attachment site at C14, 1 (6,14-AmidoHap), 2 (14-AmidoMorHap), and 3 (14-AmidoHerHap) as novel heroin haptens. The compounds 1, 2, and 3 are analogues with different substituents at C6: an acetamide, a hydroxyl moiety, and an acetate, respectively. All three haptens had a phenolic hydroxyl group at C3. The haptens were conjugated to the tetanus toxoid carrier protein, adjuvanted with liposomal monophosphoryl lipid A/aluminum hydroxide and were tested in mice in terms of immunogenicity and efficacy. Immunization of mice resulted in antibody endpoint titers of >105 against all the haptens. Neither of the conjugates of 1, 2, and 3 had induced antibodies with selectivity broad enough to recognize and bind heroin, 6-AM, and morphine resulting in little to no protection against the antinociceptive effects of heroin in vivo. Only the mice immunized with conjugate 3 were partially protected against heroin-induced antinociception. These results contribute to the growing body of knowledge that the linker position and the subtle structural differences in the hapten scaffold impact the selectivity of the induced antibodies. Together, these highlight the importance of rational hapten design for heroin vaccine development.

Effect of Preexisting Immunity to Tetanus Toxoid on the Efficacy of Tetanus Toxoid-Conjugated Heroin Vaccine in Mice.

Opioid use disorder (OUD) is a serious health problem that has dramatically increased over the last decade. Although current therapies for the management of OUD can be effective, they have limitations. The complementary strategy to combat the opioid crisis is the development of a conjugate vaccine to generate high affinity antibodies in order to neutralize opioids in circulation before reaching the brain. The components of an opioid vaccine include an opioid hapten (6-AmHap) that is conjugated to a carrier protein (tetanus toxoid) with the addition of adjuvants (Army Liposome Formulation adsorbed to aluminum hydroxide-ALFA). There is no consensus in the literature as to whether preexisting immunity to the carrier protein may impact the immunogenicity of the conjugate vaccine by inducing an enhanced or suppressed immune response to the hapten. Here, we investigated whether pre-exposure to tetanus toxoid would affect the immunogenicity and efficacy of the heroin vaccine, TT-6-AmHap. Mice were primed with diphtheria, tetanus, and acellular pertussis (DTaP) vaccine at weeks -4 and -2, then immunized with TT-6-AmHap vaccine at weeks 0, 3, and 6. Using ELISA and behavioral assays, we found that preexisting immunity to tetanus toxoid had no influence on the immunogenicity and efficacy of the TT-6-AmHap vaccine.

Orientation of Antigen Display on Self-Assembling Protein Nanoparticles Influences Immunogenicity.

Self-assembling protein nanoparticles (SAPN) serve as a repetitive antigen delivery platform with high-density epitope display; however, antigen characteristics such as size and epitope presentation can influence the immunogenicity of the assembled particle and are aspects to consider for a rationally designed effective vaccine. Here, we characterize the folding and immunogenicity of heterogeneous antigen display by integrating (a) dual-stage antigen SAPN presenting the P. falciparum (Pf) merozoite surface protein 1 subunit, PfMSP119, and Pf cell-traversal protein for ookinetes and sporozoites, PfCelTOS, in addition to (b) a homogenous antigen SAPN displaying two copies of PfCelTOS. Mice and rabbits were utilized to evaluate antigen-specific humoral and cellular induction as well as functional antibodies via growth inhibition of the blood-stage parasite. We demonstrate that antigen orientation and folding influence the elicited immune response, and when appropriately designed, SAPN can serve as an adaptable platform for an effective multi-antigen display.