RESUMEN
RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Cricetinae , Humanos , Liposomas , Ratones , Nanopartículas , ARN Mensajero , Ratas , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Distribución TisularRESUMEN
Respiratory syncytial virus (RSV) is a global public health burden for which no licensed vaccine exists. To aid vaccine development via increased understanding of the protective antibody response to RSV prefusion glycoprotein F (PreF), we performed structural and functional studies using the human neutralizing antibody (nAb) RSB1. The crystal structure of PreF complexed with RSB1 reveals a conformational, pre-fusion specific site V epitope with a unique cross-protomer binding mechanism. We identify shared structural features between nAbs RSB1 and CR9501, elucidating for the first time how diverse germlines obtained from different subjects can develop convergent molecular mechanisms for recognition of the same PreF site of vulnerability. Importantly, RSB1-like nAbs were induced upon immunization with PreF in naturally-primed cattle. Together, this work reveals new details underlying the immunogenicity of site V and further supports PreF-based vaccine development efforts.
Asunto(s)
Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Inmunogenicidad Vacunal/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Bovinos , Cristalografía por Rayos X , Humanos , Inmunización , Modelos EstructuralesRESUMEN
α-Tocopherol has been used as an immune supplement in humans, as an emulsion adjuvant component in several veterinary vaccines as well as an immunomodulatory component of AS03, an emulsion adjuvant that was used in an H1N1 pandemic vaccine (Pandemrix). AS03 is manufactured using microfluidization and high-pressure homogenization. Such high energy and complex manufacturing processes make it difficult and expensive to produce emulsion adjuvants on a large scale, especially in developing countries. In this study we have explored simpler, comparatively inexpensive methods, to formulate emulsion adjuvants containing α-tocopherol, that have the potential to be made in any well-established scale-up facility. This might facilitate producing and stock-piling adjuvant doses and therefore aide in pandemic preparedness. We used design of experiment as a tool to explore incorporating α-tocopherol into self-emulsified systems containing squalene oil and polysorbate 80. We created novel self-emulsified adjuvant systems (SE-AS) and evaluated their potency in vivo in BALB/c mice with inactivated quadrivalent influenza vaccine (QIV) and tested the cellular and humoral immune responses against the four vaccine strains.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/prevención & control , alfa-Tocoferol/administración & dosificación , Animales , Emulsiones , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Ratones , Ratones Endogámicos BALB C , Polisorbatos/química , Escualeno/química , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , alfa-Tocoferol/inmunologíaRESUMEN
The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2-20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Reacciones Antígeno-Anticuerpo , Células CHO , Cricetinae , Cricetulus , Epítopos/inmunología , Femenino , Glicosilación , Cobayas , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , Antígenos VIH/genética , Antígenos VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/prevención & control , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Polisorbatos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Escualeno/inmunologíaRESUMEN
Human respiratory syncytial virus (hRSV) is responsible for serious lower respiratory tract disease in infants and in older adults, and remains an important vaccine need. RSV fusion (F) glycoprotein is a key target for neutralizing antibodies. RSV F stabilized in its pre-fusion conformation (DS-Cav1 F) induces high neutralizing antibody titers in naïve animals, but it remains unknown to what extent pre-fusion F can boost pre-existing neutralizing responses in RSV seropositive adults. We here assess DS-Cav1 F immunogenicity in seropositive cattle pre-exposed to bovine RSV, a virus closely related to hRSV. A single immunization with non-adjuvanted DS-Cav1 F strongly boosts RSV neutralizing responses, directed towards pre-fusion F-specific epitopes, whereas a post-fusion F is unable to do so. Vaccination with pre-fusion F thus represents a promising strategy for maternal immunization and for other RSV vaccine target populations such as older adults.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Células CHO , Bovinos , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones Endogámicos BALB C , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & controlRESUMEN
Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.