A multivalent mRNA monkeypox virus vaccine (BNT166) protects mice and macaques from orthopoxvirus disease.

In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).

Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants.

The BQ and XBB subvariants of SARS-CoV-2 Omicron are now rapidly expanding, possibly due to altered antibody evasion properties deriving from their additional spike mutations. Here, we report that neutralization of BQ.1, BQ.1.1, XBB, and XBB.1 by sera from vaccinees and infected persons was markedly impaired, including sera from individuals boosted with a WA1/BA.5 bivalent mRNA vaccine. Titers against BQ and XBB subvariants were lower by 13- to 81-fold and 66- to 155-fold, respectively, far beyond what had been observed to date. Monoclonal antibodies capable of neutralizing the original Omicron variant were largely inactive against these new subvariants, and the responsible individual spike mutations were identified. These subvariants were found to have similar ACE2-binding affinities as their predecessors. Together, our findings indicate that BQ and XBB subvariants present serious threats to current COVID-19 vaccines, render inactive all authorized antibodies, and may have gained dominance in the population because of their advantage in evading antibodies.

Boosting with variant-matched or historical mRNA vaccines protects against Omicron infection in mice.

The large number of spike substitutions in Omicron lineage variants (BA.1, BA.1.1., and BA.2) could jeopardize the efficacy of SARS-CoV-2 vaccines. We evaluated in mice the protective efficacy of the Moderna mRNA-1273 vaccine against BA.1 before or after boosting. Whereas two doses of mRNA-1273 vaccine induced high levels of neutralizing antibodies against historical WA1/2020 strains, lower levels against BA.1 were associated with breakthrough infection and inflammation in the lungs. A primary vaccination series with mRNA-1273.529, an Omicron-matched vaccine, potently neutralized BA.1 but inhibited historical or other SARS-CoV-2 variants less effectively. However, boosting with either mRNA-1273 or mRNA-1273.529 vaccines increased neutralizing titers and protection against BA.1 and BA.2 infection. Nonetheless, the neutralizing antibody titers were higher, and lung viral burden and cytokines were slightly lower in mice boosted with mRNA-1273.529 and challenged with BA.1. Thus, boosting with mRNA-1273 or mRNA-1273.529 enhances protection against Omicron infection with limited differences in efficacy measured.

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

SARS-CoV-2 Omicron BA.1 breakthrough infection drives late remodeling of the memory B cell repertoire in vaccinated individuals.

How infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after SARS-CoV-2 Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of the COVID-19 mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reactions, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with a marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies.

Heavy-chain CDR3-engineered B cells facilitate in vivo evaluation of HIV-1 vaccine candidates.

V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide the maturation of other bnAb classes. To compare antigens designed to maintain this conformation, we evaluated apex-specific responses in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding the improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.

Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection.

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.

Immunodeficiency syndromes differentially impact the functional profile of SARS-CoV-2-specific T cells elicited by mRNA vaccination.

Many immunocompromised patients mount suboptimal humoral immunity after SARS-CoV-2 mRNA vaccination. Here, we assessed the single-cell profile of SARS-CoV-2-specific T cells post-mRNA vaccination in healthy individuals and patients with various forms of immunodeficiencies. Impaired vaccine-induced cell-mediated immunity was observed in many immunocompromised patients, particularly in solid-organ transplant and chronic lymphocytic leukemia patients. Notably, individuals with an inherited lack of mature B cells, i.e., X-linked agammaglobulinemia (XLA) displayed highly functional spike-specific T cell responses. Single-cell RNA-sequencing further revealed that mRNA vaccination induced a broad functional spectrum of spike-specific CD4+ and CD8+ T cells in healthy individuals and patients with XLA. These responses were founded on polyclonal repertoires of CD4+ T cells and robust expansions of oligoclonal effector-memory CD45RA+ CD8+ T cells with stem-like characteristics. Collectively, our data provide the functional continuum of SARS-CoV-2-specific T cell responses post-mRNA vaccination, highlighting that cell-mediated immunity is of variable functional quality across immunodeficiency syndromes.

Analysis of mRNA vaccination-elicited RBD-specific memory B cells reveals strong but incomplete immune escape of the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant can escape neutralization by vaccine-elicited and convalescent antibodies. Memory B cells (MBCs) represent another layer of protection against SARS-CoV-2, as they persist after infection and vaccination and improve their affinity. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant remains unclear. We assessed the affinity and neutralization potency against the Omicron variant of several hundred naturally expressed MBC-derived monoclonal IgG antibodies from vaccinated COVID-19-recovered and -naive individuals. Compared with other variants of concern, Omicron evaded recognition by a larger proportion of MBC-derived antibodies, with only 30% retaining high affinity against the Omicron RBD, and the reduction in neutralization potency was even more pronounced. Nonetheless, neutralizing MBC clones could be found in all the analyzed individuals. Therefore, despite the strong immune escape potential of the Omicron variant, these results suggest that the MBC repertoire generated by mRNA vaccines still provides some protection against the Omicron variant in vaccinated individuals.

Innate immune mechanisms of mRNA vaccines.

The lipid nanoparticle (LNP)-encapsulated, nucleoside-modified mRNA platform has been used to generate safe and effective vaccines in record time against COVID-19. Here, we review the current understanding of the manner whereby mRNA vaccines induce innate immune activation and how this contributes to protective immunity. We discuss innate immune sensing of mRNA vaccines at the cellular and intracellular levels and consider the contribution of both the mRNA and the LNP components to their immunogenicity. A key message that is emerging from recent observations is that the LNP carrier acts as a powerful adjuvant for this novel vaccine platform. In this context, we highlight important gaps in understanding and discuss how new insight into the mechanisms underlying the effectiveness of mRNA-LNP vaccines may enable tailoring mRNA and carrier molecules to develop vaccines with greater effectiveness and milder adverse events in the future.

Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination.

SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses on healthy SARS-CoV-2-naive and recovered individuals prior to and following mRNA prime and boost vaccination. Vaccination induced rapid antigen-specific CD4+ T cell responses in naive subjects after the first dose, whereas CD8+ T cell responses developed gradually and were variable in magnitude. Vaccine-induced Th1 and Tfh cell responses following the first dose correlated with post-boost CD8+ T cells and neutralizing antibodies, respectively. Integrated analysis revealed coordinated immune responses with distinct trajectories in SARS-CoV-2-naive and recovered individuals. Last, whereas booster vaccination improved T cell responses in SARS-CoV-2-naive subjects, the second dose had little effect in SARS-CoV-2-recovered individuals. These findings highlight the role of rapidly primed CD4+ T cells in coordinating responses to the second vaccine dose in SARS-CoV-2-naive individuals.

A vaccine-induced public antibody protects against SARS-CoV-2 and emerging variants.

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

COVID-19 vaccine mRNA-1273 elicits a protective immune profile in mice that is not associated with vaccine-enhanced disease upon SARS-CoV-2 challenge.

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

The potential of mRNA vaccines in cancer nanomedicine and immunotherapy.

Owing to their outstanding performance against COVID-19, mRNA vaccines have brought great hope for combating various incurable diseases, including cancer. Differences in the encoded proteins result in different molecular and cellular mechanisms of mRNA vaccines. With the rapid development of nanotechnology and molecular medicine, personalized antigen-encoding mRNA vaccines that enhance antigen presentation can trigger effective immune responses and prevent off-target toxicities. Herein, we review new insights into the influence of encoded antigens, cytokines, and other functional proteins on the mechanisms of mRNA vaccines. We also highlight the importance of delivery systems and chemical modifications for mRNA translation efficiency, stability, and targeting, and we discuss the potential problems and application prospects of mRNA vaccines as versatile tools for combating cancer.

Comb-structured mRNA vaccine tethered with short double-stranded RNA adjuvants maximizes cellular immunity for cancer treatment.

Integrating antigen-encoding mRNA (Messenger RNA) and immunostimulatory adjuvant into a single formulation is a promising approach to potentiating the efficacy of mRNA vaccines. Here, we developed a scheme based on RNA engineering to integrate adjuvancy directly into antigen-encoding mRNA strands without hampering the ability to express antigen proteins. Short double-stranded RNA (dsRNA) was designed to target retinoic acid-inducible gene-I (RIG-I), an innate immune receptor, for effective cancer vaccination and then tethered onto the mRNA strand via hybridization. Tuning the dsRNA structure and microenvironment by changing its length and sequence enabled the determination of the structure of dsRNA-tethered mRNA efficiently stimulating RIG-I. Eventually, the formulation loaded with dsRNA-tethered mRNA of the optimal structure effectively activated mouse and human dendritic cells and drove them to secrete a broad spectrum of proinflammatory cytokines without increasing the secretion of anti-inflammatory cytokines. Notably, the immunostimulating intensity was tunable by modulating the number of dsRNA along the mRNA strand, which prevents excessive immunostimulation. Versatility in the applicable formulation is a practical advantage of the dsRNA-tethered mRNA. Its formulation with three existing systems, i.e., anionic lipoplex, ionizable lipid-based lipid nanoparticles, and polyplex micelles, induced appreciable cellular immunity in the mice model. Of particular interest, dsRNA-tethered mRNA encoding ovalbumin (OVA) formulated in anionic lipoplex used in clinical trials exerted a significant therapeutic effect in the mouse lymphoma (E.G7-OVA) model. In conclusion, the system developed here provides a simple and robust platform to supply the desired intensity of immunostimulation in various formulations of mRNA cancer vaccines.

Combinatorial design of ionizable lipid nanoparticles for muscle-selective mRNA delivery with minimized off-target effects.

Ionizable lipid nanoparticles (LNPs) pivotal to the success of COVID-19 mRNA (messenger RNA) vaccines hold substantial promise for expanding the landscape of mRNA-based therapies. Nevertheless, the risk of mRNA delivery to off-target tissues highlights the necessity for LNPs with enhanced tissue selectivity. The intricate nature of biological systems and inadequate knowledge of lipid structure-activity relationships emphasize the significance of high-throughput methods to produce chemically diverse lipid libraries for mRNA delivery screening. Here, we introduce a streamlined approach for the rapid design and synthesis of combinatorial libraries of biodegradable ionizable lipids. This led to the identification of iso-A11B5C1, an ionizable lipid uniquely apt for muscle-specific mRNA delivery. It manifested high transfection efficiencies in muscle tissues, while significantly diminishing off-targeting in organs like the liver and spleen. Moreover, iso-A11B5C1 also exhibited reduced mRNA transfection potency in lymph nodes and antigen-presenting cells, prompting investigation into the influence of direct immune cell transfection via LNPs on mRNA vaccine effectiveness. In comparison with SM-102, while iso-A11B5C1's limited immune transfection attenuated its ability to elicit humoral immunity, it remained highly effective in triggering cellular immune responses after intramuscular administration, which is further corroborated by its strong therapeutic performance as cancer vaccine in a melanoma model. Collectively, our study not only enriches the high-throughput toolkit for generating tissue-specific ionizable lipids but also encourages a reassessment of prevailing paradigms in mRNA vaccine design. This study encourages rethinking of mRNA vaccine design principles, suggesting that achieving high immune cell transfection might not be the sole criterion for developing effective mRNA vaccines.