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2009 for hantaviruses Connie Schmaljohn US Army Medical Research Institute of Infectious Diseases, [email protected]

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Schmaljohn, Connie, "Vaccines for hantaviruses" (2009). US Army Research. 290. http://digitalcommons.unl.edu/usarmyresearch/290

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Vaccines for hantaviruses

Connie Schmaljohn ∗

United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, USA article info abstract

Article history: Hantaviruses are assigned to categories A and C on the National Institute of Allergy and Infectious Diseases Received 29 June 2009 (NIAID) Priority Pathogens list and to category C on the Centers for Disease Control and Prevention (CDC) Accepted 24 July 2009 Emergency Preparedness and Response list of boterrorism agents/diseases. These rodent-borne viruses are members of the family Bunyaviridae and are transmitted to humans in aerosols of rodent excreta. Keywords: There are more than 20 recognized hantaviruses, some of which are associated with one of two serious Hantavirus human diseases: hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome Hemorrhagic fever with renal syndrome (HPS). Because of the global distribution of hantaviruses, the historical association of HFRS-causing viruses Hantavirus pulmonary syndrome with military operations and the recent emergence of the HPS-causing viruses, efforts are continuing to develop safe and effective hantavirus vaccines. This review addresses the significance of hantaviruses as they relate to biodefense and provides information about conventional and molecular vaccines for HFRS that have advanced to clinical studies. Published by Elsevier Ltd.

1. Introduction the term HFRS was adopted by the World Health Organization in 1983 to consolidate the nomenclature for the diseases [4]. The anti- 1.1. Hemorrhagic fever with renal syndrome genic and molecular characterization of these viruses led to the establishment of the Hantavirus genus in the family Bunyaviridae Hantaviruses and diseases that they cause have a long associ- in 1989 [5–7]. Today, four hantaviruses are known to cause HFRS: ation with war and military operations. It is suspected that the HTNV, Seoul virus (SEOV), Puumala virus (PUUV) and Dobrava virus 7000 cases of “war nephritis” reported among British soldiers sta- (DOBV). tioned in Flanders during World War I were caused by hantavirus Not only does HFRS have a historical association with war, it is . In the early 1930s, Russian troops encountered a dis- also a significant public health threat. Thousands of cases of HFRS ease with nephritis, bleeding, and shock along the Amur River, occur in China each year, with a peak of ∼116,000 cases and ∼2500 which forms the border between far-eastern Russia and Manchuria. deaths recorded in 1986 [8,9]. The Chinese cases are caused by Around the same time, the Japanese Army suffered more than infections with either HTNV or SEOV. HFRS caused by PUUV or 12,000 cases of a similar disease when they invaded Manchuria DOBV is also highly endemic in Europe, Scandinavia, and Russia, (reviewed in [1]). Both Japanese and Chinese conducted experi- with the greatest incidences observed in Finland (∼25,000 cases ments in humans, mostly prisoners of war, and established that from 1979 to 2006) and western Russia (∼89,000 cases from 1996 to they could reproduce the disease by injecting urine or blood from 2006) [10]. Epidemics have also occurred; for example, The Repub- infected humans or field mice, and that they could serially pas- lic of Bashkortostan reported more than 10,000 cases of HFRS, with sage the disease in humans [1]. During the Korean War, a disease an incidence rate of 287 per 100,000 in 1997 [11]. characterized by shock and renal failure, known as Korean hemor- HFRS presents with sudden fever, chills, nausea, headache, and rhagic fever (KHF) or epidemic hemorrhagic fever, appeared among backache. Early symptoms of severe HFRS often also include facial several thousand United Nations personnel, leading to a quarter flushing, conjunctivitis, and petechial rash. The most serious aspect century of efforts to identify the causative agent. In the early 1980s, of the disease is vascular leakage, leading to low blood pressure, the etiologic agent of KHF, Hantaan virus (HTNV) was finally iso- acute shock, and renal failure, which can cause fluid overload. Mor- lated from the lungs of Korean field mice and propagated in cell tality ranges have been estimated from less than 1% to 15%, with culture [2,3]. Several diseases that were clinically similar to KHF HTNV and DOBV infections causing more fatalities than SEOV or were soon shown to be caused by viruses related to HTNV and PUUV infections. PUUV rarely causes shock, although renal prob- lems are common, and about 5% of patients require dialysis [12]. There is evidence that the severity of HFRS correlates with a par- ∗ ticular subset of human major histocompatibility complex (HLA), Tel.: +1 301 619 4103; fax: +1 301 619 2439. E-mail address: [email protected]. markers, suggesting a genetic susceptibility to disease [13,14].

0264-410X/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.vaccine.2009.07.096 D62 C. Schmaljohn / Vaccine 27 (2009) D61–D64

Table 1 Hantavirus Vaccines Tested in Humans.

Hantavirusa Vaccine substrate or vector Inactivation method or expression products schedule (wks) Country where tested References

HTNV Suckling mouse brain Formalin 0, 4, 52 Korea 37, 38 SEOV Golden hamster kidney cells Formalin 0, 2, 4, 52 China 34b HTNV Mongolian gerbil kidney cells ␤-propiolactone 0, 1, 2, 52 China 34b HTNV Suckling mouse brain Formalin 0, 2, 4, 52 China 34b SEOV/HTNV Golden hamster kidney cells Formalin 0, 2, 4 China 40

HTNV Vaccinia virus GN,GC,N 0, 2 U.S./Korea 42 HTNV/PUUV Plasmid pWRG7077 GN,GC 0, 4, 8 U.S. (in progress) Unpublished a Hantaan virus (HTNV); Seoul virus (SEOV); Puumala virus (PUUV). b Review includes Chinese literature references.

Complete recovery from any of these hantavirus infections can take play a role in both protective immunity and pathogenesis of the weeks or months. diseases [23,28,29]. Elucidating the exact nature of protective immune responses to 1.2. Hantavirus (cardio) pulmonary syndrome (HPS) hantaviral infections has been hindered by the lack of good ani- mal models of disease. None of the Old World hantaviruses cause The existence of hantaviruses in the US was known since 1983, frank illness or death in any animal model, although mild disease, when Prospect Hill virus (PHV) was isolated from a meadow vole similar to that sometimes observed in humans, has been reported in Frederick, MD [15,16]. Many attempts to show pathogenicity of for PUUV infections of nonhuman primates [30,31]. Similarly, all of this virus failed, and for more than a decade it was believed that the North American and most of the South American hantaviruses New World hantaviruses were not pathogenic. In addition to PHV, cause no discernible disease in animals. An exception is Andes virus rat-borne SEOV was found in several sites in the US [17], but was (ANDV), which causes a lethal disease in hamsters that is very sim- not associated with typical HFRS. SEOV, which is not native to the ilar to human HPS [32]. To date, because HPS is a relatively rare New World, was introduced when rats were brought over on ships disease, most vaccine efforts have been directed at HFRS; thus pro- from the Old World. The first appearance of an acute hantavirus- tection is usually measured by the ability to protect from . associated disease in the New World occurred in 1993 when a This review is focused only on vaccines that have been tested in cluster of cases of adult respiratory distress were observed in the humans. Other reviews have appeared recently that contain addi- southwestern US. Within days of obtaining clinical specimens, the tional sources of information on studies in animals [33–35]. disease was shown to be caused by a previously unrecognized han- tavirus, now known as Sin Nombre virus (SNV), that was carried by 2.2. Conventional vaccine approaches deer mice [18]. After the discovery of SNV, several other pathogenic and presumably nonpathogenic hantaviruses were identified in Both rodent brain and cell culture-derived inactivated vac- various rodent species throughout the New World. None of the cines for HFRS have been developed and tested in humans in North American hantaviruses have been shown to be transmit- Asia (Table 1), although none has been approved for use in the ted person-to-person; however, there is evidence that the South US. Rodent brain-derived vaccines are not common in western American virus, Andes virus, can occasionally be contagious among countries, in part because of concerns about the possibility of humans [19,20]. The disease caused by these hantaviruses, HPS, is autoimmune encephalitis due to the presence of even minute quan- sometimes referred to as hantavirus cardiopulmonary syndrome to tities of myelin basic protein contaminants [36]. A commercial recognize the cardiogenic shock often associated with the disease suckling mouse brain-derived vaccine, Hantavax®, has been mar- [21,22]. keted in Korea for more than 10 years, with no reported safety Early symptoms of HPS include fever, general malaise and mus- issues. This formalin-inactivated suckling mouse brain prepara- cle aches. In addition, nausea, vomiting, headaches, abdominal pain tion is formulated with aluminum hydroxide adjuvant and given and other symptoms can occur. After about 4–10 days, patients can as a series of two 1 month apart with a booster 12 develop a pulmonary capillary leak syndrome leading to respira- months later. The vaccine was reported to elicit antibody responses tory distress symptoms characterized by coughing and shortness detectable by ELISA in most vaccinees, but neutralizing antibod- of breath as the lungs fill with fluid. Like HFRS, HPS-severity might ies were detected in only about half of the recipients [37,38].As correlate with a genetic predisposition [23]. More than 2500 cases yet, field trials with Hantavax® have not been conducted, and of HPS have occurred in the New World since 1993 with mortality a case–control study did not have sufficient statistical power to rates ranging from 30% to 40%. demonstrate efficacy [39]. In China, HTNV- and SEOV-inactivated vaccines have been pro- 2. Vaccines for hantaviral diseases duced in Mongolian gerbil kidney or golden hamster kidney cell cultures, respectively. Comparing these vaccines and a suckling 2.1. Virus structure and immune responses mouse brain-derived HTNV vaccine in more than 100,000 humans demonstrated that the SEOV vaccine elicited neutralizing antibody Hantaviruses have three segments of negative-sense single- responses in about 80% of recipients receiving three doses, while strand RNA designated as large (L), medium (M) and small (S), three doses of either of the two HTNV vaccines elicited neutraliz- which encode the viral RNA-dependent RNA polymerase (L), enve- ing antibodies in only about half of the vaccinees (reviewed in [34]). lope glycoproteins (GN and GC), and nucleocapsid protein (N), An inactivated, bivalent vaccine was also developed and tested in respectively (reviewed in [24]). Protective immunity is generally China, which consisted of HTNV isolated from a HFRS case, and thought to correlate with neutralizing antibodies to GN and GC, SEOV isolated from a rat. The viruses were propagated in primary and passive transfer of sera containing neutralizing antibodies can hamster kidney cells, inactivated with formaldehyde, purified by provide protection against infection in animals [25–27]. Although column chromatography, and formulated with aluminum hydrox- antibody-mediated protection is probably key, cytotoxic T cells ide adjuvant. Two or three vaccinations at 2-week intervals were have been identified in samples from infected humans, and may given to more than 500 people in southern China and more than 600 C. Schmaljohn / Vaccine 27 (2009) D61–D64 D63 people in northern China. Not all vaccinees were examined for anti- not PUUV in animals [43,44]. Consequently, to protect from all body responses, but of those that were, 93% (84/90) or 92% (83/90) HFRS-causing hantaviruses, it will be necessary to include both a developed neutralizing antibodies to HTNV or SEOV, respectively HTNV (or SEOV or DOBV) and a PUUV component. Toward this goal, after three doses. The rates were only slightly lower for those that DNA vaccines for HTNV and PUUV have been developed and tested were given two doses [40]. in animals. The HTNV DNA vaccine expresses slightly modified M segments of HTNV or a human isolate of PUUV from Russia [45,46]. 2.3. Molecular vaccines A Phase 1 clinical study of these vaccines is in progress and includes three groups of nine volunteers that are receiving three Molecular approaches to vaccines for HFRS and HPS circum- doses of 8 ␮g of DNA vaccines for HTNV, PUUV, or HTNV + PUUV at vent some of the problems associated with cell culture or rodent 4-week intervals. At every dosing, each volunteer is being vacci- brain-derived vaccines. To date, only two molecular vaccines for nated at four separate sites on the inner, upper arm, with those in HFRS have been tested in humans (Table 1), the first of which was the single group receiving four administrations of HTNV or PUUV a recombinant vaccinia virus (VACV)-vectored vaccine expressing and those in the combination group receiving two each of the HTNV the M and the S segments of HTNV [41]. A Phase I dose-escalation and PUUV vaccines. The vaccines are being delivered by hand-held study was conducted in 16 volunteers divided into four groups [42]. disposable devices that propel DNA coated gold beads into the epi- One group of VACV-immune and one group of VACV-naïve indi- dermis of the volunteers (PowderMed Vaccines). This study should viduals received about 3 × 105 plaque-forming units (PFU) of the provide valuable information both about the potential of these DNA vaccine by subcutaneous (s.c.) . The other two groups vaccines, as well as about the use of the disposable gene gun devices were VACV-naïve and received about 3 × 106 or 3 × 107 PFU of vac- for eliciting safe and immunogenic responses. cine. No adverse events were observed in any of the groups, but only those receiving the highest dose developed neutralizing anti- 3. Concluding remarks bodies to HTNV, with geometric mean titers (GMT) peaking 14 days after vaccination and dropping to baseline levels at 42 days. A Hantaviruses are distributed throughout the world and cause booster vaccination, given 1-year later resulted in a rapid anemnes- serious human diseases, which pose particular threats to military tic response in the group of volunteers who had originally received personnel who might come in contact with rodents in field oper- the highest dose of vaccine. Those in the group originally receiving ations. Both conventional and molecular vaccines for HFRS have the second highest does also developed neutralizing antibodies, shown promise in clinical studies. In Asia, the focus on vaccine but the kinetics of the response were more similar to a primary development for HFRS continues to be inactivated vaccines, and response than to an anemnestic response [42]. it is likely that if these vaccines can be distributed widely enough, A second study in 12 volunteers, showed that two s.c. vacci- a continued reduction in HFRS should continue. There have been nations with the high dose elicited neutralizing antibodies in all no similar efforts for HFRS vaccine development for Europe, in volunteers, while only two of six developed neutralizing antibod- part because PUUV is so difficult to propagate in cell culture, and ies after one vaccination [42]. A final Phase 1 clinical study was rodent brain-derived vaccines are not considered desirable. More- conducted to compare s.c. inoculation to the more standard means over, because DOBV and PUUV can both cause HFRS in the same of vaccinia virus vaccination, scarification. Groups of six VACV- region, and because there is little or no cross-protective immu- immune or six VACV-naïve volunteers were vaccinated twice by nity to these hantaviruses, a comprehensive vaccine for European scarification with ∼105 PFU of the vaccine. An additional group of HFRS will require two components. A molecular vaccine, there- six VACV-immune volunteers received two s.c. with fore, appears to be a more feasible solution for HFRS prevention ∼107 PFU of vaccine. All of the scarified VACV-naïve volunteers, in Europe. but none of the VACV-immune volunteers developed neutralizing To date, vaccines for HPS have not been tested in humans. antibodies to HTNV, while four of six VACV-immune individuals Although HPS is among the most pathogenic of human viral infec- vaccinated by the s.c. route developed neutralizing antibodies to tions, it is a relatively rare disease. Consequently, funding for HPS HTNV [42]. vaccines is not a high priority for most countries. It is possible that Because the VACV-vectored vaccine also contained the N pro- as more cases are recognized, particularly in South America, vac- tein of HTNV, and because protective immunity has been associated cine efforts may receive better support. If so, molecular vaccines with responses to N in animals [25], lymphocyte proliferation would again seem to be the best approach, because producing large responses were measured for all recipients in the Phase 1 stud- quantities of an HPS virus for inactivation would require specialized ies. In general, the proliferative response paralleled the antibody high-containment facilities. Finally, although vaccines are gener- responses. Of interest, lymphocyte proliferation responses to the ally the best means to prevent any disease, it is possible to reduce HPS-causing SNV as well as to HTNV could also be detected in many HFRS and HPS infections through rodent control and through exer- of the samples, demonstrating a possible means of cross-protective cise of precautions when cleaning rodent infested areas. immunity for HFRS and HPS. 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