DNA Vaccine-Generated Duck Polyclonal Antibodies as a Postexposure Prophylactic to Prevent Hantavirus Pulmonary Syndrome (HPS)

Sin Nombre virus (SNV) and Andes virus (ANDV) cause most of the hantavirus pulmonary syndrome (HPS) cases in North and South America, respectively. The chances of a patient surviving HPS are only two in three. Previously, we demonstrated that SNV and ANDV DNA vaccines encoding the virus envelope glycoproteins elicit high-titer neutralizing antibodies in laboratory animals, and (for ANDV) in nonhuman primates (NHPs). In those studies, the vaccines were delivered by gene gun or muscle electroporation. Here, we tested whether a combined SNV/ANDV DNA vaccine (HPS DNA vaccine) could be delivered effectively using a disposable syringe jet injection (DSJI) system (PharmaJet, Inc). PharmaJet intramuscular (IM) and intradermal (ID) needle-free devices are FDA 510(k)-cleared, simple to use, and do not require electricity or pressurized gas. First, we tested the SNV DNA vaccine delivered by PharmaJet IM or ID devices in rabbits and NHPs. Both IM and ID devices produced high-titer anti-SNV neutralizing antibody responses in rabbits and NHPs. However, the ID device required at least two vaccinations in NHP to detect neutralizing antibodies in most animals, whereas all animals vaccinated once with the IM device seroconverted. Because the IM device was more effective in NHP, the Stratis® (PharmaJet IM device) was selected for follow-up studies. We evaluated the HPS DNA vaccine delivered using Stratis® and found that it produced high-titer anti-SNV and anti-ANDV neutralizing antibodies in rabbits (n=8/group) as measured by a classic plaque reduction neutralization test and a new pseudovirion neutralization assay. We were interested in determining if the differences between DSJI delivery (e.g., high-velocity liquid penetration through tissue) and other methods of vaccine injection, such as needle/syringe, might result in a more immunogenic DNA vaccine. To accomplish this, we compared the HPS DNA vaccine delivered by DSJI versus needle/syringe in NHPs (n=8/group). We found that both the anti-SNV and anti-ANDV neutralizing antibody titers were significantly higher (p-value 0.0115) in the DSJI-vaccinated groups than the needle/syringe group. For example, the anti-SNV and anti-ANDV PRNT50 geometric mean titers (GMTs) were 1,974 and 349 in the DSJI-vaccinated group versus 87 and 42 in the needle/syringe group. These data demonstrate, for the first time, that a spring-powered DSJI device is capable of effectively delivering a DNA vaccine to NHPs. Whether this HPS DNA vaccine, or any DNA vaccine, delivered by spring-powered DSJI will elicit a strong immune response in humans, requires clinical trials.

Sin Nombre virus (SNV) and Andes virus (ANDV) cause hantavirus pulmonary syndrome (HPS) in humans. Both SNV and ANDV infect Syrian hamsters, but only ANDV causes lethal disease. A co-infection study was performed to determine which virus, SNV or ANDV, would dominate the survival outcome in hamsters. Infection of hamsters with SNV 1 day before ANDV challenge did not result in disease characteristic of the latter virus, and all animals survived challenge. Control animals infected solely with ANDV all succumbed by day 14. In contrast, when viruses were injected at the same site concurrently, all hamsters succumbed to HPS disease. Hantaviruses are segmented viruses; therefore we investigated which segment might be responsible for the protective phenotype of SNV by using two SNV/ANDV reassortant viruses, both with reciprocal M-segments from the other virus (denoted ASA and SAS). Both reassortants asymptomatically infect hamsters, similar to SNV. However, unlike SNV, 1 day prior preinfection with the reassortant virus did not prevent ANDV lethality. The ASA reassortant virus, but not SAS, protected hamsters from lethal ANDV infection when administered 3 days prior to ANDV challenge. Similar to SNV preinfection, the potent innate immune stimulator poly I:C administered to hamsters 1 day before ANDV challenge prevented lethal ANDV disease. Combined, these results suggest that the difference in pathogenicity of SNV and ANDV in hamsters involves differences in early host-pathogen interactions and resultant anti-viral immune responses of both the innate and adaptive immune system.

Andes virus (ANDV) is a South American hantavirus that causes hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory infection with a case fatality rate as high as 40%. A critical model for studying HCPS is the Syrian hamster model, which faithfully recapitulates key aspects of the disease. Recent studies have shown that strains of ANDV from different origins, i.e., from rodent or human hosts, can produce widely different outcomes in the hamster model. CHI-7913, a human isolate, does not cause lethal disease and replicates poorly in hamster tissues compared with the classical rodent isolate Chile-9717869. Here, we passaged CHI-7913 in vivo in Syrian hamsters 25 times and infected groups of hamsters with virus from passages 2, 12, and 24 to assess whether viral adaptation occurs that might ultimately drive the phenotype of the virus during infection in this model. Very few mutations occurred during passage in hamsters, with only a single coding mutation discovered after 25 passages. Nearly all mutations occurring in hamsters occurred early, within two passages, and none of the passaged virus caused overt disease or pathology in infected hamsters, consistent with minimal changes in the viral genome. Early in infection, CHI-7913 viral RNA levels were still well below those seen in lethal Chile-9717869 infection. Overall, our data offer insights into the lack of selective pressure on hantaviruses in certain hosts and further evidence of distinct outcomes between rodent and human-derived viral isolates that should be studied further in additional viral species.IMPORTANCEInfection of Syrian hamsters with Andes virus (ANDV) can result in disparate outcomes, depending on the source of the virus used for the infection. The ANDV strain CHI-7913 does not cause lethal disease in hamsters but is able to replicate in hamster tissues. We successively passaged CHI-7913 in vivo to study how continued infection influences adaptation in hamsters and whether passaging would lead to the development of a lethal model, as sometimes occurs with other viruses. Surprisingly, even after 25 passages, minimal mutations occurred in ANDV CHI-7913 genomes, with only one coding mutation present above consensus by passage 25, in the viral glycoprotein. Our data suggest that depending on both viral origin and the host, hantaviruses may face minimal selective pressure to mutate toward a disease phenotype. Studies with additional ANDV strains and other hantavirus species are warranted to further study this phenomenon.

Expression and purification of the surface proteins from Andes virus

Polyclonal immunoglobulin-based medical products have been used successfully to treat diseases caused by viruses for more than a century. We demonstrate the use of DNA vaccine technology and transchromosomal bovines (TcBs) to produce fully human polyclonal immunoglobulins (IgG) with potent antiviral neutralizing activity. Specifically, two hantavirus DNA vaccines [Andes virus (ANDV) DNA vaccine and Sin Nombre virus (SNV) DNA vaccine] were used to produce a candidate immunoglobulin product for the prevention and treatment of hantavirus pulmonary syndrome (HPS). A needle-free jet injection device was used to vaccinate TcB, and high-titer neutralizing antibodies (titers >1000) against both viruses were produced within 1 month. Plasma collected at day 10 after the fourth vaccination was used to produce purified α-HPS TcB human IgG. Treatment with 20,000 neutralizing antibody units (NAU)/kg starting 5 days after challenge with ANDV protected seven of eight animals, whereas zero of eight animals treated with the same dose of normal TcB human IgG survived. Likewise, treatment with 20,000 NAU/kg starting 5 days after challenge with SNV protected immunocompromised hamsters from lethal HPS, protecting five of eight animals. Our findings that the α-HPS TcB human IgG is capable of protecting in animal models of lethal HPS when administered after exposure provides proof of concept that this approach can be used to develop candidate next-generation polyclonal immunoglobulin-based medical products without the need for human donors, despeciation protocols, or inactivated/attenuated vaccine antigen.

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

A lethal disease model for hantavirus pulmonary syndrome.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-β, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Andes virus (ANDV) selectively infects pulmonary microvascular endothelial cells (PMECs), causing a 35% fatal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). However, the mechanism of ANDV-induced vascular permeability remains an enigma. Gorbunova and Mackow (e00396-21) found that the ANDV N-protein binds to RhoGDI, the primary repressor of RhoA-directed vascular permeability. N-protein binding to S34-phosphorylated RhoGDI inhibited RhoGDI binding and repression of RhoA, revealing a novel edemagenic mechanism that permits ANDV to cause HPS. These findings rationalize targeting PKCα phosphorylation of RhoGDI to therapeutically inhibit HPS patient edema and may be broadly applicable to other causes of acute respiratory distress.

Hantaviruses are responsible for thousands of infections globally. In the absence of vaccines or targeted treatments for severe diseases such as haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome, medical care focuses solely on managing symptoms. As a result, preventive hygiene measures, such as disinfection, are essential to reduce transmission and minimize the virus's impact on human health. The aim of this study was to assess the effectiveness of commonly used disinfectants in inactivating Andes virus (ANDV) and evaluate the virus's stabilty to support evidence-based hygiene strategies. The virucidal activity of hand antiseptics against ANDV was assessed using a quantitative suspension test in accordance with the European guideline EN14476. Surface disinfection efficacy was evaluated according to EN16777 and virus stability was assessed on stainless steel. These findings indicate that ANDV has lower environmental stability compared to other enveloped viruses and can be effectively inactivated by all disinfectants tested. This data can inform risk assessments, especially in areas where hantavirus outbreaks are common, and may guide cleaning protocols for both healthcare environments and high-risk public spaces.

Since the identification of hantavirus pulmonary syndrome in the United States in 1993, Latin America has been the region of the world where more hantaviruses have been identified, associated with human disease or not. This update shows the advances in the study of hantaviruses in several countries of the region and the problems that are still unresolved. Clinical findings in Chile have shown differences in the classical description of hantavirus pulmonary syndrome in the United States. Hemorrhage was observed in 64% of the cases and alteration in renal function in 48%. The classification of cases in mild, self-limited forms and severe forms has a prognostic value, with statistically significant differences between survivors and nonsurvivors. Epidemiological studies have shown noticeable differences in seroprevalence of antibodies against hantaviruses in humans, ranging from about 1% to more than 40% according to geographical and ethnical differences. Risk factors continue to be related to rural activities and peridomestic sites. Rodent studies have allowed the identification of putative reservoirs of hantaviruses in Uruguay, Brazil and Argentina and detected antibodies in several rodent species not previously reported in Paraguay and Panama. An Andes virus gene-based hantavirus pulmonary syndrome vaccine is currently under study. Progress in research on hantaviruses has been continuous but slow. The complex picture of the viruses, the rodent reservoirs and the clinical forms of the disease mean researchers are faced with the great challenge of properly clarifying the genetic and pathogenic relationships between hantaviruses in the Americas.

Cases of Hantavirus cardiopulmonary syndrome (HCPS) have been reported in Chile between Region V and XI. In our country HCPS is caused by Andes virus. Hantavirus infection is acquired mainly through inhalation of aerosolized rodent fluids. Person to person transmission is considered exceptional. The identified rodent reservoir of the virus extends throughout Region III to XII, and no cases have been reported in the north of Chile. The incubation period is prolonged. We report the first diagnosed patient with HCPS in the north of Chile. In this case we suggest the possibility of person to person transmission.

Hantavirus is associated with hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The clinical diagnosis of hantavirus infections has been confirmed routinely by the immunofluorescence antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA). A rapid and easy diagnostic test for hantavirus infection is required. A new immunochromatographic assay for hantavirus, POC-PUU, useful for the diagnosis of epidemic nephrophaty associated with hantavirus Puumala in Europe, was evaluated in Chile. This test is based on recombinant N-protein of hantavirus Puumala, and cross-reacts with other hantaviruses. Eighty human sera were selected at random from patients from Southern Chile who were suspected with HPS. The hantavirus capture-IgM ELISA was compared with a commercially available POC-PUU test (POC PUUMALA, Reagena Ltd., Toivala, Finland). The test sensitivity and specificity of the POC-PUU test were 97 and 90%, respectively. It is important to note that although the test is not specific for Andes virus the sensitivity and specificity were above 90%, which indicates good reactivity to the Puumala nucleoprotein antigen. As this test is cost-effective, with a high negative value, rapid and easy to carry out, specialized personnel are not necessary, nor does it require specialized equipment. Its usefulness for diagnosis is important in hospitals far from reference centers and areas with a high incidence of HPS cases.

Nosocomial transmission of Andes virus has been documented in Argentina, but has not yet been proven in Chile. We studied 215 contacts (106 family member contacts and 109 health care worker contacts) of 20 index cases of hantavirus cardiopulmonary syndrome (HCPS) in Chile. The seroprevalence of IgG antibodies against Andes virus was 1.9% (95% confidence interval [CI] = 0.34-6.3%) among the family members and 0.0% (95% CI = 0-3.2%) among the health care workers. Our data suggest that there is no evidence for nosocomial transmission of Andes virus in region IX of Chile.

Hantaviridae currently encompasses seven genera and 53 species. Multiple hantaviruses such as Hantaan virus, Seoul virus, Dobrava-Belgrade virus, Puumala virus, Andes virus, and Sin Nombre virus are highly pathogenic to humans. They cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome or hantavirus pulmonary syndrome (HCPS/HPS) in many countries. Some hantaviruses infect wild or domestic animals without causing severe symptoms. Rodents, shrews, and bats are reservoirs of various mammalian hantaviruses. Recent years have witnessed significant advancements in the study of hantaviruses including genomics, taxonomy, evolution, replication, transmission, pathogenicity, control, and patient treatment. Additionally, new hantaviruses infecting bats, rodents, shrews, amphibians, and fish have been identified. This review compiles these advancements to aid researchers and the public in better recognizing this zoonotic virus family with global public health significance.