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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-SIV CT.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.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the Omicron lineage has resulted in diminished Coronavirus Disease 2019 (COVID-19) vaccine efficacy and persistent transmission. In this study, we evaluated the immunogenicity and protective efficacy of two, recently authorized, bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary two-dose immunization series in mice, both bivalent vaccines induced greater neutralizing antibody responses against Omicron variants than the parental, monovalent mRNA-1273 vaccine. When administered to mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced broadly neutralizing antibody responses. Whereas most anti-Omicron receptor binding domain antibodies in serum induced by mRNA-1273, mRNA-1273.214 and mRNA-1273.222 boosters cross-reacted with the antecedent Wuhan-1 spike antigen, the mRNA-1273.214 and mRNA-1273.222 bivalent vaccine boosters also induced unique BA.1-specific and BA.4/5-specific responses, respectively. Although boosting with parental or bivalent mRNA vaccines substantially improved protection against BA.5 compared to mice receiving two vaccine doses, the levels of infection, inflammation and pathology in the lung were lowest in animals administered the bivalent mRNA vaccines. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and confers protection in mice against a currently circulating SARS-CoV-2 strain., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, Senda Biosciences, and Carnival Corporation, and on the scientific advisory boards of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from J. Virol. Biotechnol., Kaleido, and Emergent BioSolutions and past support from Moderna not related to these studies. K.W., D.L., L.E.A., L.M., T.K., C.S., A.W., A.C., S.M.E., G.-Y.C., G.S.-J., I.R., A.M., Y.-T.L., and D.K.E. are employees of and shareholders in Moderna., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 ID 50 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., Competing Interests: Declaration of interests K.S.C. is an inventor on U.S. Patent no. 10,960,070 B2 and International Patent Application no. WO/2018/081318 entitled “Prefusion Coronavirus Spike Proteins and Their Use.” K.S.C. is an inventor on U.S. Patent Application no. 62/972,886 entitled “2019-nCoV Vaccine.” A.R.H., L.W., W.S., Y.Z., E.S.Y., J.R.M., P.D.K., M.R., N.J.S., and D.C.D. are inventors on U.S. Patent Application no. 63/147,419 entitled “Antibodies Targeting the Spike Protein of Coronaviruses.” L.P., A.V.R., B.N., D.V., A. Cook, A.D., K.S., H.A., and M.G.L. are employees of Bioqual. K.S.C., L.W., W.S., and Y.Z. are inventors on multiple U.S. Patent Applications entitled “Anti-Coronavirus Antibodies and Methods of Use.” G.-Y.C., G.S.-J., I.R., Y.-T.L., A.M., K.W., A. Carfi, and D.K.E. are employees of Moderna. M.S.S. serves on the scientific board of advisors for Moderna and Ocugen. The other authors declare no competing interests., (Published by Elsevier Inc.)

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., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

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 immunogenicity 1 . 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 mutant 2 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.

Chimeric antigen receptors (CARs) are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA). Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN) lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro , and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO-), DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

Although peptides are well recognised biological molecules in vivo, their selection from libraries is challenging because of relative low affinity whilst in linear conformation. We hypothesized that multiplexed peptides and DNA on the surface of beads would provide a platform for enhanced avidity and the selection of relevant peptides from a library (ORBIT bead display). Using human immunodeficiency virus (HIV-1) gp120 as a target, we identify peptides that inhibit HIV-1 replication in vitro through blocking of protein:protein interaction with the co-receptor CCR5. The bead display approach has many potential applications for probing biological systems and for drug lead development.

Background: Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant., Methods: Based on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments., Results: It was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region., Conclusions: Our results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).

The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evolution, and cross-species reactivity has been suggested to be a common feature of different animals based on research in humans and mice. However, we found that CD1d from the tree shrew (Tupaia belangeri), a close evolutionary relative of primates, failed to stimulate human iNKT cells, despite being more homologous to human CD1d than that of mouse. Sequence comparison and molecular modelling showed that two of the key amino acid residues in human CD1d proposed to be in direct contact with T-cell receptors were mutated in tree shrew CD1d. Substitution of one of the residues, but not the other, with the human residue enabled tree shrew CD1d to regain the ability to present lipid antigen to human iNKT cells. These results indicate that CD1d/iNKT recognition is species-specific, and that cross-species reactivity may be less common than currently proposed. Also, a naturally occurring CD1d mutation(s) that confers inability to stimulate iNKT cell function may have implications for future studies on CD1d/iNKT-associated diseases.

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.

HIV-specific cytotoxic T lymphocytes (CTL) are important in controlling HIV replication, but the magnitude of the CTL response does not predict clinical outcome. In four donors with delayed disease progression we identified Vbeta13.2 T cell receptors (TCRs) with very similar and unusually long beta-chain complementarity determining region 3 (CDR3) regions in CTL specific for the immunodominant human histocompatibility leukocyte antigens (HLA)-B8-restricted human immunodeficiency virus-1 (HIV-1) nef epitope, FLKEKGGL (FL8). CTL expressing Vbeta13.2 TCRs tolerate naturally arising viral variants in the FL8 epitope that escape recognition by other CTL. In addition, they expand efficiently in vitro and are resistant to apoptosis, in contrast to FL8-specific CTL using other TCRs. Selection of Vbeta13.2 TCRs by some patients early in the FL8-specific CTL response may be linked with better clinical outcome.

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2-restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR-peptide-major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR-peptide-MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes.