Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all vaccine candidates. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence and four key changes conserved between immunogens. The changes are adaptable to all vaccine platforms, are compatible with established changes in SARS-CoV-2 vaccines, and are compatible with mutations in emerging variants of concern. The immunogens elicit higher levels of neutralizing antibodies than native RBD, focus the immune response to structured neutralizing epitopes, and have increased production yields and thermostability. Incorporating these variant-independent amino acid changes in next-generation vaccines may enhance the neutralizing antibody response and lead to pan-SARS-CoV-2 protection.

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Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation

Journal of Virology ◽

10.1128/jvi.02437-16 ◽

2017 ◽

Vol 91 (13) ◽

Cited By ~ 18

Author(s):

Normand Blais ◽

Martin Gagné ◽

Yoshitomo Hamuro ◽

Patrick Rheault ◽

Martine Boyer ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Antibody Response ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Human Respiratory Syncytial Virus ◽

Vaccine Antigen ◽

Significant Advance ◽

F Protein ◽

Syncytial Virus

ABSTRACT The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen. IMPORTANCE Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.

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Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800224115 ◽

2018 ◽

Vol 115 (24) ◽

pp. 6273-6278 ◽

Cited By ~ 53

Author(s):

Ilona Baraniak ◽

Barbara Kropff ◽

Lyn Ambrose ◽

Megan McIntosh ◽

Gary R. McLean ◽

...

Keyword(s):

Antibody Response ◽

Neutralizing Antibodies ◽

De Novo ◽

Natural Infection ◽

Neutralizing Antibody ◽

Glycoprotein B ◽

Solid Organ ◽

Viral Glycoprotein ◽

Viral Spread

Human cytomegalovirus (HCMV) is an important pathogen in transplant patients and in congenital infection. Previously, we demonstrated that vaccination with a recombinant viral glycoprotein B (gB)/MF59 adjuvant formulation before solid organ transplant reduced viral load parameters post transplant. Reduced posttransplant viremia was directly correlated with antibody titers against gB consistent with a humoral response against gB being important. Here we show that sera from the vaccinated seronegative patients displayed little evidence of a neutralizing antibody response against cell-free HCMV in vitro. Additionally, sera from seronegative vaccine recipients had minimal effect on the replication of a strain of HCMV engineered to be cell-associated in a viral spread assay. Furthermore, although natural infection can induce antibody-dependent cellular cytotoxicity (ADCC) responses, serological analysis of seronegative vaccinees again presented no evidence of a substantial ADCC-promoting antibody response being generated de novo. Finally, analyses for responses against major antigenic domains of gB following vaccination were variable, and their pattern was distinct compared with natural infection. Taken together, these data argue that the protective effect elicited by the gB vaccine is via a mechanism of action in seronegative vaccinees that cannot be explained by neutralization or the induction of ADCC. More generally, these data, which are derived from a human challenge model that demonstrated that the gB vaccine is protective, highlight the need for more sophisticated analyses of new HCMV vaccines over and above the quantification of an ability to induce potent neutralizing antibody responses in vitro.

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Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518405113 ◽

2016 ◽

Vol 113 (16) ◽

pp. 4446-4451 ◽

Cited By ~ 13

Author(s):

Jordan R. Willis ◽

Jessica A. Finn ◽

Bryan Briney ◽

Gopal Sapparapu ◽

Vidisha Singh ◽

...

Keyword(s):

Amino Acid ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Computational Design ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Amino Acid Length ◽

Massive Scale ◽

Amino Acid Mutations ◽

Hiv 1

Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1–naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1–naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 107unique HCDR3 amino acid regions from 70 different HIV-1–naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1–naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts.

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Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.80.8.3994-4004.2006 ◽

2006 ◽

Vol 80 (8) ◽

pp. 3994-4004 ◽

Cited By ~ 183

Author(s):

Israrul H. Ansari ◽

Byungjoon Kwon ◽

Fernando A. Osorio ◽

Asit K. Pattnaik

Keyword(s):

Amino Acid ◽

Neutralizing Antibodies ◽

Protective Efficacy ◽

Neutralizing Antibody ◽

Respiratory Syndrome Virus ◽

Virus Infectivity ◽

Neutralization Epitope ◽

Enhanced Sensitivity

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.

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A prevaccination validated network that drives the breadth of the protective neutralizing antibody response following Dengue Vaccine TV003 immunization

10.1101/2021.09.25.21264123 ◽

2021 ◽

Author(s):

Adam-Nicolas Pelletier ◽

Gabriela Pacheco Sanchez ◽

Mark Watson ◽

Abdullah Izmirly ◽

Tiziana Di Pucchio ◽

...

Keyword(s):

Immune Response ◽

Antibody Response ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Inflammasome Activation ◽

Proinflammatory Response ◽

Protective Antibody ◽

Secondary Bile Acids

Development of fully protective dengue virus (DV) vaccines has been problematic as infection with DV requires a broad antibody immune response that targets all 4 possible serotypes. Herein, we used an integrated systems vaccinology approach to identify prevaccination features that allow the development of fully protective DV-specific antibody responses. This approach allowed us to identify a transcription network in a subset of monocytes defined by the expression of CD68 and downstream of specific pro- and anti-inflammatory cytokines. Moreover, we identified metabolites as drivers of an immune response that induced neutralizing antibodies to the 4 DV serotypes. Specifically, PC/PE drove the production of TGF-B in CD68 low monocytes, which was a positive correlate of the protective antibody response. In contrast, primary and secondary bile acids triggered a proinflammatory response downstream of TGR5 signaling and inflammasome activation in CD68 high monocytes, which was associated to a non-protective antibody response. These features were validated in vitro in primary myeloid cells. Our results highlight the role of cell and systemic metabolism as regulators of protective immune responses to vaccination, and that systems vaccinology is a key tool to identify such mechanisms.

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Neutralizing Antibody Responses to Human Herpesviruses 6 and 7 Do Not Cross-React with Each Other, and Maternal Neutralizing Antibodies Contribute to Sequential Infection with These Viruses in Childhood

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.2.388-393.2002 ◽

2002 ◽

Vol 9 (2) ◽

pp. 388-393 ◽

Cited By ~ 3

Author(s):

Mariko Yoshida ◽

Sadayoshi Torigoe ◽

Kumiko Ikeue ◽

Masao Yamada

Keyword(s):

Antibody Response ◽

Neutralizing Antibodies ◽

Human Herpesvirus ◽

Neutralizing Antibody ◽

Young Group ◽

Adult Group ◽

Dot Blot ◽

Serum Samples ◽

Antibody Titers ◽

Almost All

ABSTRACT Seroprevalence of human herpesvirus 6 (HHV-6) and HHV-7 infections is very high throughout the world, and almost all people are exposed first to HHV-6 and second to HHV-7 in their childhood. However, it is not clear whether the neutralizing (NT) antibody response between each virus is cross-reactive or not. To elucidate the NT antibody response between each virus, 55 serum samples from an adult group (subjects 22 to 88 years old) and 60 serum samples from a young group (subjects 2 to 18 years old) were examined by a dot blot method for detecting viral late antigen. Thirty-nine serum samples obtained from cord bloods and a few serum samples obtained from pediatric patients with exanthem subitum were also examined to assess the maternal transferred NT antibodies against each virus. The NT antibody titers against HHV-7 in the adult group remained high throughout all the individuals, and none were negative. Those against HHV-6 were high values in the young group but low values, including negative values (three samples), in the adult group. These results suggested that the NT antibody response to either HHV-6 or HHV-7 in each individual was specific to each virus and did not cross-react with each other. In the adult group, the NT antibody response to HHV-6 decreased, while that to HHV-7 remained high throughout all the individuals. Maternal transferred NT antibody titers against HHV-7 were higher and remained longer after birth than those of HHV-6, and these findings were in accord with the clinical observation that HHV-6 infection usually occurs earlier than HHV-7 infection.

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In vitro neutralizing activity of BNT162b2 mRNA-induced antibodies against full B.1.351 SARS-CoV-2 variant

10.22541/au.163881397.76967489/v1 ◽

2021 ◽

Author(s):

Federico García ◽

Esther Serrano-Conde ◽

Alba Leyva ◽

Ana Fuentes-Lopez ◽

Adolfo de Salazar ◽

...

Keyword(s):

Single Dose ◽

Antibody Response ◽

Neutralizing Antibodies ◽

Healthcare Workers ◽

Neutralizing Antibody ◽

Neutralization Activity ◽

Neutralizing Activity ◽

Study Participants ◽

Prior Infection

Background: SARS-CoV-2 variation represents a serious challenge to current COVID-19 vaccines. Recent reports suggest that B.1.351 and other variants may escape the neutralization activity of the antibodies generated by current vaccines. Methods: Ninety-nine healthcare workers undertaking BNT162b2 mRNA vaccination were sampled at baseline, on the day of the second dose, and 14 days after the latter. Neutralization activity against SARS-CoV-2 B.1, B.1.1.7 and B.1.351 was investigated using a Vero-E6 model. Results: Eleven of the study participants had prior infection with SARS-CoV-2. Neutralization titers against the B.1 and the B.1.1.7 variants were not statistically different and were significantly higher than titers against the B.1.351 variant across pre-exposed and non-pre-exposed vaccinated individuals ( p<0.01). While all vaccinated individuals presented neutralizing antibodies against B.1 and B 1.1.7 after the second dose, 14% were negative against B.1.351, and 76% had low titers (1/20-1/80). Pre-exposed vaccinated individuals showed higher titers than non-pre-exposed after the first (median titers of 1/387 versus 1/28, respectively) and the second doses (1/995 versus 1/703, respectively). As high as 72% of the pre-exposed vaccinees presented titers >1/80 after a single dose, while only 11% of non-exposed vaccinated individuals had titers >1/80. Conclusions: BNT162b2 mRNA-induced antibodies show a lower in vitro neutralizing activity against B.1.351 variant compared to neutralization against B.1.1.7 or B.1 variants. Interestingly, for individuals pre-exposed to SARS-CoV-2, one dose of BNT162b2 mRNA may be adequate to produce neutralizing antibodies against B.1.1.7 and B.1, while two doses of BNT162b2 mRNA provide optimal neutralizing antibody response against B.1.351 too.

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The quantitative landscape of the neutralizing antibody response to SARS-CoV-2

10.1101/2020.09.25.20201996 ◽

2020 ◽

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra M Dixit

Keyword(s):

Clinical Trials ◽

Antibody Response ◽

Dose Response ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Virtual Patient ◽

Plasma Samples ◽

Response Curves

Neutralizing antibodies (NAbs) appear promising interventions against SARS-CoV-2 infection. Over 100 NAbs have been identified so far and several are in clinical trials. Yet, which NAbs would be the most potent remains unclear. Here, we analysed reported in vitro dose-response curves (DRCs) of >70 NAbs and estimated corresponding 50% inhibitory concentrations, slope parameters, and instantaneous inhibitory potentials (IIPs), presenting a comprehensive quantitative landscape of NAb responses to SARS-CoV-2. NAbs with high IIPs are likely to be potent. To assess the applicability of the landscape in vivo, we analysed available DRCs of NAbs from individual patients and found that the responses closely resembled the landscape. Further, we created virtual patient plasma samples by randomly sampling NAbs from the landscape and found that they recapitulated plasma dilution assays from convalescent patients. The landscape thus offers a facile tool for benchmarking NAbs and would aid the development of NAb-based therapies for SARS-CoV-2 infection.

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SARS-CoV-2 RBD trimer protein adjuvanted with Alum-3M-052 protects from SARS-CoV-2 infection and immune pathology in the lung

Nature Communications ◽

10.1038/s41467-021-23942-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nanda Kishore Routhu ◽

Narayanaiah Cheedarla ◽

Venkata Satish Bollimpelli ◽

Sailaja Gangadhara ◽

Venkata Viswanadh Edara ◽

...

Keyword(s):

Antibody Response ◽

Antibody Titer ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Neutralizing Antibody ◽

Significant Loss ◽

Lung Pathology ◽

Live Virus ◽

Suitable Candidate ◽

Immune Pathology

AbstractThere is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1μg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2.

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Virus-Neutralizing Monoclonal Antibody Expressed in Milk of Transgenic Mice Provides Full Protection against Virus-Induced Encephalitis

Journal of Virology ◽

10.1128/jvi.75.6.2803-2809.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2803-2809 ◽

Cited By ~ 21

Author(s):

Andreas F. Kolb ◽

Lecia Pewe ◽

John Webster ◽

Stanley Perlman ◽

C. Bruce A. Whitelaw ◽

...

Keyword(s):

Transgenic Mice ◽

Neutralizing Antibodies ◽

Defense Mechanism ◽

Viral Infections ◽

Hepatitis Virus ◽

Recombinant Antibody ◽

Lethal Dose ◽

Neutralizing Antibody ◽

Murine Hepatitis Virus

ABSTRACT Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine β-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.

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