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).

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.

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.

Malaria transmission-blocking conjugate vaccine in ALFQ adjuvant induces durable functional immune responses in rhesus macaques.

Malaria transmission-blocking vaccines candidates based on Pfs25 and Pfs230 have advanced to clinical studies. Exoprotein A (EPA) conjugate of Pfs25 in Alhydrogel® developed functional immunity in humans, with limited durability. Pfs230 conjugated to EPA (Pfs230D1-EPA) with liposomal adjuvant AS01 is currently in clinical trials in Mali. Studies with these conjugates revealed that non-human primates are better than mice to recapitulate the human immunogenicity and functional activity. Here, we evaluated the effect of ALFQ, a liposomal adjuvant consisting of TLR4 agonist and QS21, on the immunogenicity of Pfs25-EPA and Pfs230D1-EPA in Rhesus macaques. Both conjugates generated strong antibody responses and functional activity after two vaccinations though activity declined rapidly. A third vaccination of Pfs230D1-EPA induced functional activity lasting at least 9 months. Antibody avidity increased with each vaccination and correlated strongly with functional activity. IgG subclass analysis showed induction of Th1 and Th2 subclass antibody levels that correlated with activity.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

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.

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.

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.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of other coronaviruses. Here, we designed and characterized four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion Spike (S), S1 and RBD. These immunogens induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2 in mice. A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers more than 20-fold higher than convalescent donor serum, following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 virus challenge. Furthermore, SpFN- and RFN-immunization elicited ACE2 blocking activity and neutralizing ID50 antibody titers >2,000 against SARS-CoV-1, along with high magnitude neutralizing titers against major VoC. These results provide design strategies for pan-coronavirus vaccine development. HIGHLIGHTS: Iterative structure-based design of four Spike-domain Ferritin nanoparticle classes of immunogensSpFN-ALFQ and RFN-ALFQ immunization elicits potent neutralizing activity against SARS-CoV-2, variants of concern, and SARS-CoV-1Passively transferred IgG from immunized C57BL/6 mice protects K18-hACE2 mice from lethal SARS-CoV-2 challenge.

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.

Design of Alphavirus Virus-Like Particles Presenting Circumsporozoite Junctional Epitopes That Elicit Protection against Malaria.

The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope-CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.

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.

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.

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.

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.

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.

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.

Army Liposome Formulation (ALF) family of vaccine adjuvants.

Introduction: From its earliest days, the US. military has embraced the use of vaccines to fight infectious diseases. The Army Liposome Formulation (ALF) has been a pivotal innovation as a vaccine adjuvant that provides excellent safety and potency and could lead to dual-use military and civilian benefits. For protection of personnel against difficult disease threats found in many areas of the world, Army vaccine scientists have created novel liposome-based vaccine adjuvants.Areas covered: ALF consists of liposomes containing saturated phospholipids, cholesterol, and monophosphoryl lipid A (MPLA) as an immunostimulant. ALF exhibited safety and strong potency in many vaccine clinical trials. Improvements based on ALF include: ALF adsorbed to aluminum hydroxide (ALFA); ALF containing QS21 saponin (ALFQ); and ALFQ adsorbed to aluminum hydroxide (ALFQA). Preclinical safety and efficacy studies with ALF, LFA, ALFQ, and ALFQA are discussed in preparation for upcoming vaccine trials targeting malaria, HIV-1, bacterial diarrhea, and opioid addiction.Expert opinion: The introduction of ALF in the 1980s stimulated commercial interest in vaccines to infectious diseases, and therapeutic vaccines to cancer, and Alzheimer's disease. It is likely that ALF, ALFA, and ALFQ, will provide momentum for new types of modern vaccines with improved efficacy and safety.

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.