Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies
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eLife assessment
Antibodies perform a critical function in host defense against viruses and have emerged as major therapeutic tools in modern medicine, as evidenced by the large scale use antibody-based therapies during the COVID-19 pandemic. This paper describes the characterization of human antibodies to hantaviruses that have the potential to create devastating epidemics. The results teach us about the viral structures that are targets for neutralization and the results are relevant for vaccine development and antibody therapeutic design.
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Abstract
Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While infections in humans are relatively rare, mortality rates range from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the human humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity to authentic hantaviruses. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines.
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Author Response
Reviewer #1 (Public Review):
This work focuses on the characterization of neutralizing antibodies from humans survivors of SNV and ANDV hantavirus infections, including the mapping of epitopes located in the Gn and/or Gc glycoproteins, and their mechanism of viral interference blocking receptor binding or membrane fusion. It also confirms previous data on broadly neutralizing epitopes allowing inhibition of different hantavirus species. The work covers for the first time in vivo evidence of cross-protection against HNTV infection by a broadly neutralizing antibody prepared from SNV infection using a prophylaxis animal model and compares the data with protection from ANDV lethal challenge using ANDV-specific neutralizing antibodies. The work provides valuable information for the development of therapeutic measures that …
Author Response
Reviewer #1 (Public Review):
This work focuses on the characterization of neutralizing antibodies from humans survivors of SNV and ANDV hantavirus infections, including the mapping of epitopes located in the Gn and/or Gc glycoproteins, and their mechanism of viral interference blocking receptor binding or membrane fusion. It also confirms previous data on broadly neutralizing epitopes allowing inhibition of different hantavirus species. The work covers for the first time in vivo evidence of cross-protection against HNTV infection by a broadly neutralizing antibody prepared from SNV infection using a prophylaxis animal model and compares the data with protection from ANDV lethal challenge using ANDV-specific neutralizing antibodies. The work provides valuable information for the development of therapeutic measures that cross-protect against several hantavirus species which seems a promising strategy to rise pharmaceutical interest against a group of viruses causing orphan disease.
The strength of the work is based on the impressive amount of work and versatility of methods to identify residues involved in the binding and/or escape from seven different neutralizing antibody clones that allow for important conclusions on species-specific antigenic regions and confirm data on a region that seems broadly conserved among different hantavirus species. At the same time, the weakness of the work is that data processing does not allow for readers data analysis (Figs. 1b, 2a, 2c, Ext. Data Fig. 4).
The authors clearly achieve their aim of characterizing the antigenic sites of neutralizing antibodies. Yet, the presented data on binding to ANDV mutant constructs and negative-staining EM does not allow for the conclusion that the epitope of the broadly neutralizing antibodies ANDV-44 and SNV-53 involved the Gn capping loop. An alternative explanation of the escape mutations in the Gn capping loop could be produced by an allosteric effect on the Gc fusion loop region, and a role in structuring the Gc fusion loop has been previously demonstrated (References 7 and 9). In addition, it is not clear why SNV-24 has no broad neutralizing activity although escape mutations occurred at the highly conserved residues K833 and D822 in Gc domain I.
. . . it would be important to show viral RNA levels in lungs and kidneys in the lethal ANDV animal model (Fig. 7) to allow for comparison with the prophylaxis from HTNV infection (Fig. 6).
ANDV does not necessarily cause significant viremia but this challenge model does allow detection of substantial virus load in organs. To monitor virus in organs, a separate animal study would be required with serial euthanasia. All treated animals survived and were kept until day 28. The previous study (DOI: 10.1016/j.celrep.2021.109086) demonstrated that virus was not detected in animals that survived until day 28. Here, we would have to perform another ABSL3 animal experiment with euthanasia and harvest organs at the expected peak for viral replication to confirm this finding. We do not believe repeating such a study is justified at this point, since the key endpoint for the experiment here is survival, and the study provided clear results. Increasing the number of animals in study in order to euthanize a subset in order to collect organs on a specific day makes more sense in a drug discovery effort where a candidate drug is not expected to protect the animals but might have some impact on the virologic endpoint only (e.g., reduce viremia in blood or organs). Thus, we do not believe repeated studies are justified to obtain this additional confirmatory data point.
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eLife assessment
Antibodies perform a critical function in host defense against viruses and have emerged as major therapeutic tools in modern medicine, as evidenced by the large scale use antibody-based therapies during the COVID-19 pandemic. This paper describes the characterization of human antibodies to hantaviruses that have the potential to create devastating epidemics. The results teach us about the viral structures that are targets for neutralization and the results are relevant for vaccine development and antibody therapeutic design.
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Reviewer #1 (Public Review):
This work focuses on the characterization of neutralizing antibodies from humans survivors of SNV and ANDV hantavirus infections, including the mapping of epitopes located in the Gn and/or Gc glycoproteins, and their mechanism of viral interference blocking receptor binding or membrane fusion. It also confirms previous data on broadly neutralizing epitopes allowing inhibition of different hantavirus species. The work covers for the first time in vivo evidence of cross-protection against HNTV infection by a broadly neutralizing antibody prepared from SNV infection using a prophylaxis animal model and compares the data with protection from ANDV lethal challenge using ANDV-specific neutralizing antibodies. The work provides valuable information for the development of therapeutic measures that cross-protect …
Reviewer #1 (Public Review):
This work focuses on the characterization of neutralizing antibodies from humans survivors of SNV and ANDV hantavirus infections, including the mapping of epitopes located in the Gn and/or Gc glycoproteins, and their mechanism of viral interference blocking receptor binding or membrane fusion. It also confirms previous data on broadly neutralizing epitopes allowing inhibition of different hantavirus species. The work covers for the first time in vivo evidence of cross-protection against HNTV infection by a broadly neutralizing antibody prepared from SNV infection using a prophylaxis animal model and compares the data with protection from ANDV lethal challenge using ANDV-specific neutralizing antibodies. The work provides valuable information for the development of therapeutic measures that cross-protect against several hantavirus species which seems a promising strategy to rise pharmaceutical interest against a group of viruses causing orphan disease.
The strength of the work is based on the impressive amount of work and versatility of methods to identify residues involved in the binding and/or escape from seven different neutralizing antibody clones that allow for important conclusions on species-specific antigenic regions and confirm data on a region that seems broadly conserved among different hantavirus species. At the same time, the weakness of the work is that data processing does not allow for readers data analysis (Figs. 1b, 2a, 2c, Ext. Data Fig. 4).
The authors clearly achieve their aim of characterizing the antigenic sites of neutralizing antibodies. Yet, the presented data on binding to ANDV mutant constructs and negative-staining EM does not allow for the conclusion that the epitope of the broadly neutralizing antibodies ANDV-44 and SNV-53 involved the Gn capping loop. An alternative explanation of the escape mutations in the Gn capping loop could be produced by an allosteric effect on the Gc fusion loop region, and a role in structuring the Gc fusion loop has been previously demonstrated (References 7 and 9). In addition, it is not clear why SNV-24 has no broad neutralizing activity although escape mutations occurred at the highly conserved residues K833 and D822 in Gc domain I.
Finally, concerning the in vivo protection experiments, it would be important to show viral RNA levels in lungs and kidneys in the lethal ANDV animal model (Fig. 7) to allow for comparison with the prophylaxis from HTNV infection (Fig. 6).
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Reviewer #2 (Public Review):
Treatment of human illnesses caused by infection by hantaviruses are currently not available and hence research on new therapies are needed. The manuscript by Engdahl et al describes the characterization of four neutralizing antibodies with potency against hantaviruses using several approaches. This knowledge of these antibodies and where they bind in these studies can be used in the design of vaccines or the development of passive immunotherapeutic approaches and are hence very valuable for the advancement of new treatments. Hence this new knowledge is a major strength of the manuscript. the studies, however, the in vitro studies are limited in the use of pseudotyped viruses and not the actual viruses. Inclusion of the potency and binding of these to their native viruses, and standardization of their use in …
Reviewer #2 (Public Review):
Treatment of human illnesses caused by infection by hantaviruses are currently not available and hence research on new therapies are needed. The manuscript by Engdahl et al describes the characterization of four neutralizing antibodies with potency against hantaviruses using several approaches. This knowledge of these antibodies and where they bind in these studies can be used in the design of vaccines or the development of passive immunotherapeutic approaches and are hence very valuable for the advancement of new treatments. Hence this new knowledge is a major strength of the manuscript. the studies, however, the in vitro studies are limited in the use of pseudotyped viruses and not the actual viruses. Inclusion of the potency and binding of these to their native viruses, and standardization of their use in treatments of hamsters with these viruses, would elevate this approach to stand as a valuable contribution to the development of treatments for hantaviruses.
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Reviewer #3 (Public Review):
In this manuscript, the authors characterize antigen binding sites, mechanism of action, and in vivo efficacy of neutralizing monoclonal antibodies (mAbs) previously isolated from New World hantavirus survivors. Both hantavirus species-specific mAbs and broadly neutralizing hantavirus mAbs are analyzed.
The strengths of the manuscript are the presentation of both in vitro and in vivo data for mAbs that have different antigen binding sites and mechanisms of neutralization. Weaknesses include a lack of authentic virus experiments for the in vitro data.
The impact of the work on the field is the identification of different neutralizing sites on hantavirus glycoproteins in species-specific and broadly reactive mAbs. There are also interesting data on loss of broadly neutralizing activity of mAbs after reversion …
Reviewer #3 (Public Review):
In this manuscript, the authors characterize antigen binding sites, mechanism of action, and in vivo efficacy of neutralizing monoclonal antibodies (mAbs) previously isolated from New World hantavirus survivors. Both hantavirus species-specific mAbs and broadly neutralizing hantavirus mAbs are analyzed.
The strengths of the manuscript are the presentation of both in vitro and in vivo data for mAbs that have different antigen binding sites and mechanisms of neutralization. Weaknesses include a lack of authentic virus experiments for the in vitro data.
The impact of the work on the field is the identification of different neutralizing sites on hantavirus glycoproteins in species-specific and broadly reactive mAbs. There are also interesting data on loss of broadly neutralizing activity of mAbs after reversion to the germline sequence.
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