Multivalent mRNA-DTP vaccines are immunogenic and provide protection from Bordetella pertussis challenge in mice.

Acellular multivalent vaccines for pertussis (DTaP and Tdap) prevent symptomatic disease and infant mortality, but immunity to Bordetella pertussis infection wanes significantly over time resulting in cyclic epidemics of pertussis. The messenger RNA (mRNA) vaccine platform provides an opportunity to address complex bacterial infections with an adaptable approach providing Th1-biased responses. In this study, immunogenicity and challenge models were used to evaluate the mRNA platform with multivalent vaccine formulations targeting both B. pertussis antigens and diphtheria and tetanus toxoids. Immunization with mRNA formulations were immunogenetic, induced antigen specific antibodies, as well as Th1 T cell responses. Upon challenge with either historical or contemporary B. pertussis strains, 6 and 10 valent mRNA DTP vaccine provided protection equal to that of 1/20th human doses of either DTaP or whole cell pertussis vaccines. mRNA DTP immunized mice were also protected from pertussis toxin challenge as measured by prevention of lymphocytosis and leukocytosis. Collectively these pre-clinical mouse studies illustrate the potential of the mRNA platform for multivalent bacterial pathogen vaccines.

mRNA-LNP prime boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Divergent antibody recognition profiles are generated by protective mRNA vaccines against Marburg and Ravn viruses.

The first-ever recent Marburg virus (MARV) outbreak in Ghana, West Africa and Equatorial Guinea has refocused efforts towards the development of therapeutics since no vaccine or treatment has been approved. mRNA vaccines were proven successful in a pandemic-response to severe acute respiratory syndrome coronavirus-2, making it an appealing vaccine platform to target highly pathogenic emerging viruses. Here, 1-methyl-pseudouridine-modified mRNA vaccines formulated in lipid nanoparticles (LNP) were developed against MARV and the closely-related Ravn virus (RAVV), which were based on sequences of the glycoproteins (GP) of the two viruses. Vaccination of guinea pigs with both vaccines elicited robust binding and neutralizing antibodies and conferred complete protection against virus replication, disease and death. The study characterized antibody responses to identify disparities in the binding and functional profiles between the two viruses and regions in GP that are broadly reactive. For the first time, the glycan cap is highlighted as an immunoreactive site for marburgviruses, inducing both binding and neutralizing antibody responses that are dependent on the virus. Profiling the antibody responses against the two viruses provided an insight into how antigenic differences may affect the response towards conserved GP regions which would otherwise be predicted to be cross-reactive and has implications for the future design of broadly protective vaccines. The results support the use of mRNA-LNPs against pathogens of high consequence.

An mRNA-based platform for the delivery of pathogen-specific IgA into mucosal secretions.

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions.

A Lassa virus mRNA vaccine confers protection but does not require neutralizing antibody in a guinea pig model of infection.

Lassa virus is a member of the Arenaviridae family, which causes human infections ranging from asymptomatic to severe hemorrhagic disease with a high case fatality rate. We have designed and generated lipid nanoparticle encapsulated, modified mRNA vaccines that encode for the wild-type Lassa virus strain Josiah glycoprotein complex or the prefusion stabilized conformation of the Lassa virus glycoprotein complex. Hartley guinea pigs were vaccinated with two 10 µg doses, 28 days apart, of either construct. Vaccination induced strong binding antibody responses, specific to the prefusion conformation of glycoprotein complex, which were significantly higher in the prefusion stabilized glycoprotein complex construct group and displayed strong Fc-mediated effects. However, Lassa virus-neutralizing antibody activity was detected in some but not all animals. Following the challenge with a lethal dose of the Lassa virus, all vaccinated animals were protected from death and severe disease. Although the definitive mechanism of protection is still unknown, and assessment of the cell-mediated immune response was not investigated in this study, these data demonstrate the promise of mRNA as a vaccine platform against the Lassa virus and that protection against Lassa virus can be achieved in the absence of virus-neutralizing antibodies.

Increased neutralization potency and breadth elicited by a SARS-CoV-2 mRNA vaccine forming virus-like particles.

Vaccines have played a fundamental role in the control of infectious diseases. We previously developed a messenger RNA (mRNA) vaccine against HIV-1 that forms virus-like particles (VLPs) through coexpression of the viral envelope with Gag. Here, we applied the same principle to the design of a VLP-forming mRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To promote cognate interaction with simian immunodeficiency virus (SIV) Gag, we engineered different chimeric proteins encompassing the ectodomain and the transmembrane region of the SARS-CoV-2 Spike protein from the Wuhan-Hu-1 strain fused to the gp41 cytoplasmic tail of either HIV-1 (strain WITO) or SIV (strain mac239) with or without a partial truncation at amino acid 745 to enhance membrane expression. Upon cotransfection with SIV gag mRNA, the Spike-SIVCT.745 (SSt) chimera yielded the highest level of cell-surface expression and extracellular VLP release. Immunization of BALB/c mice with SSt+gag mRNA at 0, 4, and 16 wk induced higher titers of Spike-binding and autologous neutralizing antibodies at all time points compared to SSt mRNA alone. Furthermore, mice immunized with SSt+gag mRNA developed neutralizing antibodies effective against different variants of concern. These data demonstrate that the Gag/VLP mRNA platform can be successfully applied to vaccines against different agents for the prevention of infectious diseases of global relevance.

Rational Design and In Vivo Characterization of mRNA-Encoded Broadly Neutralizing Antibody Combinations against HIV-1.

Monoclonal antibodies have been used successfully as recombinant protein therapy; however, for HIV, multiple broadly neutralizing antibodies may be necessary. We used the mRNA-LNP platform for in vivo co-expression of 3 broadly neutralizing antibodies, PGDM1400, PGT121, and N6, directed against the HIV-1 envelope protein. mRNA-encoded HIV-1 antibodies were engineered as single-chain Fc (scFv-Fc) to overcome heavy- and light-chain mismatch. In vitro neutralization breadth and potency of the constructs were compared to their parental IgG form. We assessed the ability of these scFv-Fcs to be expressed individually and in combination in vivo, and neutralization and pharmacokinetics were compared to the corresponding full-length IgGs. Single-chain PGDM1400 and PGT121 exhibited neutralization potency comparable to parental IgG, achieving peak systemic concentrations ≥ 30.81 µg/mL in mice; full-length N6 IgG achieved a peak concentration of 974 µg/mL, but did not tolerate single-chain conversion. The mRNA combination encoding full-length N6 IgG and single-chain PGDM1400 and PGT121 was efficiently expressed in mice, achieving high systemic concentration and desired neutralization potency. Analysis of mice sera demonstrated each antibody contributed towards neutralization of multiple HIV-1 pseudoviruses. Together, these data show that the mRNA-LNP platform provides a promising approach for antibody-based HIV treatment and is well-suited for development of combination therapeutics.

Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.

Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice.

Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.

A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus.

Chikungunya virus (CHIKV) infection causes acute disease characterized by fever, rash and arthralgia, which progresses to severe and chronic arthritis in up to 50% of patients. Moreover, CHIKV infection can be fatal in infants or immunocompromised individuals and has no approved therapy or prevention. This phase 1, first-in-human, randomized, placebo-controlled, proof-of-concept trial conducted from January 2019 to June 2020 evaluated the safety and pharmacology of mRNA-1944, a lipid nanoparticle-encapsulated messenger RNA encoding the heavy and light chains of a CHIKV-specific monoclonal neutralizing antibody, CHKV-24 ( NCT03829384 ). The primary outcome was to evaluate the safety and tolerability of escalating doses of mRNA-1944 administered via intravenous infusion in healthy participants aged 18-50 years. The secondary objectives included determination of the pharmacokinetics of mRNA encoding for CHKV-24 immunoglobulin heavy and light chains and ionizable amino lipid component and the pharmacodynamics of mRNA-1944 as assessed by serum concentrations of mRNA encoding for CHKV-24 immunoglobulin G (IgG), plasma concentrations of ionizable amino lipid and serum concentrations of CHKV-24 IgG. Here we report the results of a prespecified interim analysis of 38 healthy participants who received intravenous single doses of mRNA-1944 or placebo at 0.1, 0.3 and 0.6 mg kg-1, or two weekly doses at 0.3 mg kg-1. At 12, 24 and 48 h after single infusions, dose-dependent levels of CHKV-24 IgG with neutralizing activity were observed at titers predicted to be therapeutically relevant concentrations (≥1 µg ml-1) across doses that persisted for ≥16 weeks at 0.3 and 0.6 mg kg-1 (mean t1/2 approximately 69 d). A second 0.3 mg kg-1 dose 1 week after the first increased CHKV-24 IgG levels 1.8-fold. Adverse effects were mild to moderate in severity, did not worsen with a second mRNA-1944 dose and none were serious. To our knowledge, mRNA-1944 is the first mRNA-encoded monoclonal antibody showing in vivo expression and detectable ex vivo neutralizing activity in a clinical trial and may offer a treatment option for CHIKV infection. Further evaluation of the potential therapeutic use of mRNA-1944 in clinical trials for the treatment of CHIKV infection is warranted.

A multiclade env-gag VLP mRNA vaccine elicits tier-2 HIV-1-neutralizing antibodies and reduces the risk of heterologous SHIV infection in macaques.

The development of a protective vaccine remains a top priority for the control of the HIV/AIDS pandemic. Here, we show that a messenger RNA (mRNA) vaccine co-expressing membrane-anchored HIV-1 envelope (Env) and simian immunodeficiency virus (SIV) Gag proteins to generate virus-like particles (VLPs) induces antibodies capable of broad neutralization and reduces the risk of infection in rhesus macaques. In mice, immunization with co-formulated env and gag mRNAs was superior to env mRNA alone in inducing neutralizing antibodies. Macaques were primed with a transmitted-founder clade-B env mRNA lacking the N276 glycan, followed by multiple booster immunizations with glycan-repaired autologous and subsequently bivalent heterologous envs (clades A and C). This regimen was highly immunogenic and elicited neutralizing antibodies against the most prevalent (tier-2) HIV-1 strains accompanied by robust anti-Env CD4+ T cell responses. Vaccinated animals had a 79% per-exposure risk reduction upon repeated low-dose mucosal challenges with heterologous tier-2 simian-human immunodeficiency virus (SHIV AD8). Thus, the multiclade env-gag VLP mRNA platform represents a promising approach for the development of an HIV-1 vaccine.

Chimeric Fusion (F) and Attachment (G) Glycoprotein Antigen Delivery by mRNA as a Candidate Nipah Vaccine.

Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.

Broadly neutralizing monoclonal antibodies protect against multiple tick-borne flaviviruses.

Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type-specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses.

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.

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.