Maternally Derived Recombinant Human Anti-Hantavir
http://www.100md.com
病菌学杂志 2006年第8期
State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100094
State Key Laboratory for Infectious Diseases Control and Prevention, China CDC, Beijing 100052
Animal Science and Technology College of He Bei Agricultural University, Baoding 071001, People'sRepublic of China
ABSTRACT
Transgenic mice expressing a recombinant human monoclonal antibody (rHMAb) against hantavirus were generated. These mice could be used as models to explore the possibilities of producing rHMAbs for therapeutic purposes. The highest concentration of the rHMAb in the milk of the transgenic females was 6.6 mg/ml. The rHMAb was also detected in the sera of pups fed by the transgenic females. Both the rHMAbs in the milk of transgenic mice and those in the sera of suckling pups were found to be active against hantaviruses, although the light chain of the antibody absorbed by the pups was modified by N-linked glycosylation.
TEXT
Hantaviruses compose a genus that belongs to the family Bunyaviridae but differ from the viruses of other genera in that they are enzootic (24). The natural hosts of hantaviruses are rodents, and transmission from their natural hosts to humans is not well known but it is generally accepted that inhalation of infected excreta causes infection (20, 29). Some species of hantaviruses cause severe infections in humans such as hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (8, 22). The annually reported number of cases of hemorrhagic fever with renal syndrome caused by hantaviruses is about 150,000 worldwide, and two-thirds of the cases occur in China and are mainly associated with Hantaan (HTNV) and Seoul (SEOV) hantavirus infections (26).
To prevent hantavirus infection, vaccines consisting of inactivated viruses (formalin-inactivated, rodent brain-derived virus) (5, 28) have been developed. Recently, vaccines made by recombinant DNA technology (including recombinant vaccinia virus and naked DNA vaccines) have also been shown to be promising (6, 9, 14, 18). Protective immunity to hantavirus infections has previously been associated with neutralizing antibody responses directed against the viral G1 and G2 envelope glycoproteins (1, 2). High concentrations of neutralizing antibodies in serum efficiently block infection (12). However, production of sufficient quantities of monoclonal antibodies (MAbs) for therapy remains a major problem (12, 15). Production of MAbs in the milk of transgenic animals is one of the most attractive techniques for addressing this problem (10, 11).
In this study, the heavy-chain and light-chain genes of a human immunoglobulin G1 (IgG1) MAb against the HTNV G2 protein (12) were cloned into a commercial pBC1 vector and co-microinjected to create transgenic mice expressing a recombinant human MAb (rHMAb) in their milk. Altogether, 75 mice were produced through co-microinjection. PCR and Southern blotting identified seven (two females and five males) transgenic founders containing both the heavy- and light-chain genes (Fig. 1).
High levels of rHMAbs directed against hantavirus were detected in the milk (but not the serum) of the founder (hAHT5) and the F1 females (hAHT8-6, hAHT12-20, hAHT61-28, hAHT71-38, and hAHT71-64) (Fig. 2). Expression levels of the recombinant antibody in the milk of F0 and F1 transgenic (both heavy- and light-chain-containing) females were more than 1 mg/ml, and 6.6 mg/ml was the highest expression level found (Table 1). One F1 mouse, hAHT8-9, showed no intact antibody in the milk when tested by enzyme-linked immunosorbent assay, despite a strong positive signal for both the heavy- and light-chain antibodies in its whey by Western blotting. Also, we found that the hAHT44 mouse, which had acquired only the heavy-chain gene, did not express detectable levels of the heavy-chain antibody.
The activity of rHMAbs was determined by using immunofluorescent antibody (IFA) (Fig. 3). The results showed that expressed rHMAbs, exemplified by one of the transgenic whey samples and one of the serum samples collected from the offspring of transgenic females, could both bind specifically to the G2 antigen of HTNV. As the MAb against the HTNV G2 protein is able to bind both HTNV and SEOV, additional testing for the binding of rHMAbs to SEOV antigen slides was also performed. The results showed that expressed rHMAbs could bind to both HTNV and SEOV. The rHMAbs in the serum from the pups also showed binding to HTNV and SEOV antigens despite modifications (discussed below).
To test if rHMAbs in the transgenic milk could be absorbed by pups and provide protection against HTNV, sera were collected from the 5-day-old offspring of two transgenic mice expressing high levels of rHMAbs against hantavirus (hAHT12-20 and hAHT61-28). Western blotting showed that the human IgG MAb was present in the sera of pups, regardless of whether they were transgenic, as long as they were suckled by transgenic mice (Fig. 4). As expected, rHMAbs were not found in the sera of the nontransgenic pups that were fed by nontransgenic mothers, indicating that rHMAbs in milk are absorbed directly by newborn pups.
The activity of rHMAbs in the milk of the transgenic females and in the sera of pups suckling transgenic mothers was further determined based on their ability to neutralize hantaviruses (Tables 1 and 2). In the neutralization test, dilutions of transgenic whey or pups' sera were added to HTNV before challenging Vero-E6 cells with the virus. Neutralized HTNV cannot infect Vero-E6 cells, and dilutions were considered to have virus-neutralizing activity if less than 50% of the challenged cells were infected by HTNV as detected by IFA. The results (Tables 1 and 2), demonstrating the neutralizing activity of the rHMAbs toward hantaviruses, suggest that protection may be provided to the offspring of transgenic animals via milk.
Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight of the rHMAb heavy-chain antibody in the sera of pups was lower than that found in the controls (rHMAb from the milk of the hAHT5 mouse and human IgG), while that of the light-chain antibody was higher (Fig. 5). These changes in molecular weight are likely to be due to glycosylations or other modifications. To validate this hypothesis, the pups' sera were digested with N-glycosidase and then analyzed by SDS-PAGE and Western blotting (Fig. 5). After enzymatic treatment, the molecular weight of the light chain antibodies in the pups' serum became lower while that of the heavy chain antibodies remained constant. Control proteins did not show any changes, suggesting that the light chain of rHMAb absorbed by the pups is modified by N-linked glycosylation. To test the change in molecular weight of the heavy-chain antibody, commercial human IgG was added to both the serum of the nontransgenic mice and the serum of the transgenic pups. This was then analyzed by SDS-PAGE and Western blotting. We found that the heavy-chain band of standard human IgG was consistent with the reduced size found for the recombinant antibody in the pups' sera, suggesting that the apparent changes in the heavy-chain antibody in sera from the pups are due to interference by other proteins and impurities.
We have achieved expression levels of up to 6.6 mg/ml of recombinant antibody in the milk of transgenic females, which compares favorably with values (0.4 to 6 mg/ml) reported previously (4, 7, 13, 16, 21, 25, 27). In vivo protection studies should have been done, but a lack of containment and strict regulations regarding animal experiments dealing with disease-causing viruses after a severe acute respiratory syndrome outbreak in China have prevented us from challenging transgenic animals or their offspring with viruses. It has previously been shown that administration of neutralizing MAbs can protect animals from infection with hantaviruses (23). The neutralization activity of the rHMAb detected in the sera of pups implies that the maternally derived recombinant antibodies may protect newborns, which are usually more susceptible to viral infections than older animals are, from death, as it has been reported that hantavirus infections are age dependent (19). It is worth noting that hantavirus infection of humans appears to be mediated by inflammatory immune responses (17, 22) and antibody treatment may potentially strengthen the inflammation. IgM antibodies are generally proinflammatory since they are powerful activators of the complement system (3). However, anti-inflammatory properties of antibodies, particularly some IgGs, have also been observed in diverse models of infectious diseases (3). Future production of the rHMAb in the milk of large transgenic animals such as cows or goats may provide sufficient antibodies for therapeutic use in humans.
ACKNOWLEDGMENTS
This work was supported by the 863 High Technology Program of the Chinese National Foundation.
We are grateful to Lennart Hammarstrom, Kasper Krogh-Anderson, Neha Pant, and Yaofeng Zhao (Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden) for reading the manuscript.
REFERENCES
Arikawa, J., A. L. Schmaljohn, J. M. Dalrymple, and C. S. Schmaljohn. 1989. Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies. J. Gen. Virol. 70(Pt. 3):615-624.
Arikawa, J., J. S. Yao, K. Yoshimatsu, I. Takashima, and N. Hashimoto. 1992. Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies. Arch. Virol. 126:271-281.
Casadevall, A., and L. A. Pirofski. 2004. New concepts in antibody-mediated immunity. Infect. Immun. 72:6191-6196.
Castilla, J., B. Pintado, I. Sola, J. M. Sanchez-Morgado, and L. Enjuanes. 1998. Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk. Nat. Biotechnol. 16:349-354.
Cho, H. W., and C. R. Howard. 1999. Antibody responses in humans to an inactivated hantavirus vaccine (Hantavax). Vaccine 17:2569-2575.
Custer, D. M., E. Thompson, C. S. Schmaljohn, T. G. Ksiazek, and J. W. Hooper. 2003. Active and passive vaccination against hantavirus pulmonary syndrome with Andes virus M genome segment-based DNA vaccine. J. Virol. 77:9894-9905.
Gavin, W. G., P. D. Pollock, D. Fell, C. Yelton, M. Cammuso, J. Harrington, P. Lewis-Williams, A. Midura, T. E. Oliver, B. Smith, Y. Wilburn, and A. H. M. Echelard. 1997. Expression of the antibody hBR96-2 in the milk of transgenic mice and production of hBR96-2 transgenic goats. Theriogenology 47:214.
Hjelle, B., S. A. Jenison, D. E. Goade, W. B. Green, R. M. Feddersen, and A. A. Scott. 1995. Hantaviruses: clinical, microbiologic, and epidemiologic aspects. Crit. Rev. Clin. Lab. Sci. 32:469-508.
Hooper, J. W., K. I. Kamrud, F. Elgh, D. Custer, and C. S. Schmaljohn. 1999. DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against Seoul virus infection. Virology 255:269-278.
Houdebine, L. M. 2002. Antibody manufacture in transgenic animals and comparisons with other systems. Curr. Opin. Biotechnol. 13:625-629.
Humphreys, D. P., and D. J. Glover. 2001. Therapeutic antibody production technologies: molecules, applications, expression and purification. Curr. Opin. Drug Discov. Dev. 4:172-185.
Koch, J., M. Liang, I. Queitsch, A. A. Kraus, and E. K. Bautz. 2003. Human recombinant neutralizing antibodies against Hantaan virus G2 protein. Virology 308:64-73.
Kolb, A. F., L. Pewe, J. Webster, S. Perlman, C. B. Whitelaw, and S. G. Siddell. 2001. Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis. J. Virol. 75:2803-2809.
Koletzki, D., R. Schirmbeck, A. Lundkvist, H. Meisel, D. H. Kruger, and R. Ulrich. 2001. DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection. J. Biotechnol. 84:73-78.
Liang, M., M. Mahler, J. Koch, Y. Ji, D. Li, C. Schmaljohn, and E. K. Bautz. 2003. Generation of an HFRS patient-derived neutralizing recombinant antibody to Hantaan virus G1 protein and definition of the neutralizing domain. J. Med. Virol. 69:99-107.
Limonta, J., A. Pedraza, A. Rodriguez, F. M. Freyre, A. M. Barral, F. O. Castro, R. Lleonart, C. A. Gracia, J. V. Gavilondo, and J. de la Fuente. 1995. Production of active anti-CD6 mouse/human chimeric antibodies in the milk of transgenic mice. Immunotechnology 1:107-113.
Maes, P., J. Clement, I. Gavrilovskaya, and M. Van Ranst. 2004. Hantaviruses: immunology, treatment, and prevention. Viral Immunol. 17:481-497.
McClain, D. J., P. L. Summers, S. A. Harrison, A. L. Schmaljohn, and C. S. Schmaljohn. 2000. Clinical evaluation of a vaccinia-vectored Hantaan virus vaccine. J. Med. Virol. 60:77-85.
Nakamura, T., R. Yanagihara, C. J. Gibbs, Jr., H. L. Amyx, and D. C. Gajdusek. 1985. Differential susceptibility and resistance of immunocompetent and immunodeficient mice to fatal Hantaan virus infection. Arch. Virol. 86:109-120.
Netski, D., B. H. Thran, and S. C. St. Jeor. 1999. Sin Nombre virus pathogenesis in Peromyscus maniculatus. J. Virol. 73:585-591.
Newton, D. L., D. Pollock, P. DiTullio, Y. Echelard, M. Harvey, B. Wilburn, J. Williams, H. R. Hoogenboom, J. C. Raus, H. M. Meade, and S. M. Rybak. 1999. Antitransferrin receptor antibody-RNase fusion protein expressed in the mammary gland of transgenic mice. J. Immunol. Methods 231:159-167.
Peters, C. J., G. L. Simpson, and H. Levy. 1999. Spectrum of hantavirus infection: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Annu. Rev. Med. 50:531-545.
Schmaljohn, C. S., Y. K. Chu, A. L. Schmaljohn, and J. M. Dalrymple. 1990. Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants. J. Virol. 64:3162-3170.
Schmaljohn, C. S., and J. M. Dalrymple. 1983. Analysis of Hantaan virus RNA: evidence for a new genus of Bunyaviridae. Virology 131:482-491.
Sola, I., J. Castilla, B. Pintado, J. M. Sanchez-Morgado, C. B. Whitelaw, A. J. Clark, and L. Enjuanes. 1998. Transgenic mice secreting coronavirus neutralizing antibodies into the milk. J. Virol. 72:3762-3772.
Song, G. 1999. Epidemiological progresses of hemorrhagic fever with renal syndrome in China. Chin. Med. J. (Engl. Ed.) 112:472-477.
van Kuik-Romeijn, P., N. de Groot, E. Hooijberg, and H. A. de Boer. 2000. Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice. Transgenic Res. 9:155-159.
Yamanishi, K., O. Tanishita, M. Tamura, H. Asada, K. Kondo, M. Takagi, I. Yoshida, T. Konobe, and K. Fukai. 1988. Development of inactivated vaccine against virus causing haemorrhagic fever with renal syndrome. Vaccine 6:278-282.
Yanagihara, R., H. L. Amyx, and D. C. Gajdusek. 1985. Experimental infection with Puumala virus, the etiologic agent of nephropathia epidemica, in bank voles (Clethrionomys glareolus). J. Virol. 55:34-38.(Shuyang Yu, Mifang Liang,)
State Key Laboratory for Infectious Diseases Control and Prevention, China CDC, Beijing 100052
Animal Science and Technology College of He Bei Agricultural University, Baoding 071001, People'sRepublic of China
ABSTRACT
Transgenic mice expressing a recombinant human monoclonal antibody (rHMAb) against hantavirus were generated. These mice could be used as models to explore the possibilities of producing rHMAbs for therapeutic purposes. The highest concentration of the rHMAb in the milk of the transgenic females was 6.6 mg/ml. The rHMAb was also detected in the sera of pups fed by the transgenic females. Both the rHMAbs in the milk of transgenic mice and those in the sera of suckling pups were found to be active against hantaviruses, although the light chain of the antibody absorbed by the pups was modified by N-linked glycosylation.
TEXT
Hantaviruses compose a genus that belongs to the family Bunyaviridae but differ from the viruses of other genera in that they are enzootic (24). The natural hosts of hantaviruses are rodents, and transmission from their natural hosts to humans is not well known but it is generally accepted that inhalation of infected excreta causes infection (20, 29). Some species of hantaviruses cause severe infections in humans such as hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (8, 22). The annually reported number of cases of hemorrhagic fever with renal syndrome caused by hantaviruses is about 150,000 worldwide, and two-thirds of the cases occur in China and are mainly associated with Hantaan (HTNV) and Seoul (SEOV) hantavirus infections (26).
To prevent hantavirus infection, vaccines consisting of inactivated viruses (formalin-inactivated, rodent brain-derived virus) (5, 28) have been developed. Recently, vaccines made by recombinant DNA technology (including recombinant vaccinia virus and naked DNA vaccines) have also been shown to be promising (6, 9, 14, 18). Protective immunity to hantavirus infections has previously been associated with neutralizing antibody responses directed against the viral G1 and G2 envelope glycoproteins (1, 2). High concentrations of neutralizing antibodies in serum efficiently block infection (12). However, production of sufficient quantities of monoclonal antibodies (MAbs) for therapy remains a major problem (12, 15). Production of MAbs in the milk of transgenic animals is one of the most attractive techniques for addressing this problem (10, 11).
In this study, the heavy-chain and light-chain genes of a human immunoglobulin G1 (IgG1) MAb against the HTNV G2 protein (12) were cloned into a commercial pBC1 vector and co-microinjected to create transgenic mice expressing a recombinant human MAb (rHMAb) in their milk. Altogether, 75 mice were produced through co-microinjection. PCR and Southern blotting identified seven (two females and five males) transgenic founders containing both the heavy- and light-chain genes (Fig. 1).
High levels of rHMAbs directed against hantavirus were detected in the milk (but not the serum) of the founder (hAHT5) and the F1 females (hAHT8-6, hAHT12-20, hAHT61-28, hAHT71-38, and hAHT71-64) (Fig. 2). Expression levels of the recombinant antibody in the milk of F0 and F1 transgenic (both heavy- and light-chain-containing) females were more than 1 mg/ml, and 6.6 mg/ml was the highest expression level found (Table 1). One F1 mouse, hAHT8-9, showed no intact antibody in the milk when tested by enzyme-linked immunosorbent assay, despite a strong positive signal for both the heavy- and light-chain antibodies in its whey by Western blotting. Also, we found that the hAHT44 mouse, which had acquired only the heavy-chain gene, did not express detectable levels of the heavy-chain antibody.
The activity of rHMAbs was determined by using immunofluorescent antibody (IFA) (Fig. 3). The results showed that expressed rHMAbs, exemplified by one of the transgenic whey samples and one of the serum samples collected from the offspring of transgenic females, could both bind specifically to the G2 antigen of HTNV. As the MAb against the HTNV G2 protein is able to bind both HTNV and SEOV, additional testing for the binding of rHMAbs to SEOV antigen slides was also performed. The results showed that expressed rHMAbs could bind to both HTNV and SEOV. The rHMAbs in the serum from the pups also showed binding to HTNV and SEOV antigens despite modifications (discussed below).
To test if rHMAbs in the transgenic milk could be absorbed by pups and provide protection against HTNV, sera were collected from the 5-day-old offspring of two transgenic mice expressing high levels of rHMAbs against hantavirus (hAHT12-20 and hAHT61-28). Western blotting showed that the human IgG MAb was present in the sera of pups, regardless of whether they were transgenic, as long as they were suckled by transgenic mice (Fig. 4). As expected, rHMAbs were not found in the sera of the nontransgenic pups that were fed by nontransgenic mothers, indicating that rHMAbs in milk are absorbed directly by newborn pups.
The activity of rHMAbs in the milk of the transgenic females and in the sera of pups suckling transgenic mothers was further determined based on their ability to neutralize hantaviruses (Tables 1 and 2). In the neutralization test, dilutions of transgenic whey or pups' sera were added to HTNV before challenging Vero-E6 cells with the virus. Neutralized HTNV cannot infect Vero-E6 cells, and dilutions were considered to have virus-neutralizing activity if less than 50% of the challenged cells were infected by HTNV as detected by IFA. The results (Tables 1 and 2), demonstrating the neutralizing activity of the rHMAbs toward hantaviruses, suggest that protection may be provided to the offspring of transgenic animals via milk.
Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight of the rHMAb heavy-chain antibody in the sera of pups was lower than that found in the controls (rHMAb from the milk of the hAHT5 mouse and human IgG), while that of the light-chain antibody was higher (Fig. 5). These changes in molecular weight are likely to be due to glycosylations or other modifications. To validate this hypothesis, the pups' sera were digested with N-glycosidase and then analyzed by SDS-PAGE and Western blotting (Fig. 5). After enzymatic treatment, the molecular weight of the light chain antibodies in the pups' serum became lower while that of the heavy chain antibodies remained constant. Control proteins did not show any changes, suggesting that the light chain of rHMAb absorbed by the pups is modified by N-linked glycosylation. To test the change in molecular weight of the heavy-chain antibody, commercial human IgG was added to both the serum of the nontransgenic mice and the serum of the transgenic pups. This was then analyzed by SDS-PAGE and Western blotting. We found that the heavy-chain band of standard human IgG was consistent with the reduced size found for the recombinant antibody in the pups' sera, suggesting that the apparent changes in the heavy-chain antibody in sera from the pups are due to interference by other proteins and impurities.
We have achieved expression levels of up to 6.6 mg/ml of recombinant antibody in the milk of transgenic females, which compares favorably with values (0.4 to 6 mg/ml) reported previously (4, 7, 13, 16, 21, 25, 27). In vivo protection studies should have been done, but a lack of containment and strict regulations regarding animal experiments dealing with disease-causing viruses after a severe acute respiratory syndrome outbreak in China have prevented us from challenging transgenic animals or their offspring with viruses. It has previously been shown that administration of neutralizing MAbs can protect animals from infection with hantaviruses (23). The neutralization activity of the rHMAb detected in the sera of pups implies that the maternally derived recombinant antibodies may protect newborns, which are usually more susceptible to viral infections than older animals are, from death, as it has been reported that hantavirus infections are age dependent (19). It is worth noting that hantavirus infection of humans appears to be mediated by inflammatory immune responses (17, 22) and antibody treatment may potentially strengthen the inflammation. IgM antibodies are generally proinflammatory since they are powerful activators of the complement system (3). However, anti-inflammatory properties of antibodies, particularly some IgGs, have also been observed in diverse models of infectious diseases (3). Future production of the rHMAb in the milk of large transgenic animals such as cows or goats may provide sufficient antibodies for therapeutic use in humans.
ACKNOWLEDGMENTS
This work was supported by the 863 High Technology Program of the Chinese National Foundation.
We are grateful to Lennart Hammarstrom, Kasper Krogh-Anderson, Neha Pant, and Yaofeng Zhao (Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden) for reading the manuscript.
REFERENCES
Arikawa, J., A. L. Schmaljohn, J. M. Dalrymple, and C. S. Schmaljohn. 1989. Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies. J. Gen. Virol. 70(Pt. 3):615-624.
Arikawa, J., J. S. Yao, K. Yoshimatsu, I. Takashima, and N. Hashimoto. 1992. Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies. Arch. Virol. 126:271-281.
Casadevall, A., and L. A. Pirofski. 2004. New concepts in antibody-mediated immunity. Infect. Immun. 72:6191-6196.
Castilla, J., B. Pintado, I. Sola, J. M. Sanchez-Morgado, and L. Enjuanes. 1998. Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk. Nat. Biotechnol. 16:349-354.
Cho, H. W., and C. R. Howard. 1999. Antibody responses in humans to an inactivated hantavirus vaccine (Hantavax). Vaccine 17:2569-2575.
Custer, D. M., E. Thompson, C. S. Schmaljohn, T. G. Ksiazek, and J. W. Hooper. 2003. Active and passive vaccination against hantavirus pulmonary syndrome with Andes virus M genome segment-based DNA vaccine. J. Virol. 77:9894-9905.
Gavin, W. G., P. D. Pollock, D. Fell, C. Yelton, M. Cammuso, J. Harrington, P. Lewis-Williams, A. Midura, T. E. Oliver, B. Smith, Y. Wilburn, and A. H. M. Echelard. 1997. Expression of the antibody hBR96-2 in the milk of transgenic mice and production of hBR96-2 transgenic goats. Theriogenology 47:214.
Hjelle, B., S. A. Jenison, D. E. Goade, W. B. Green, R. M. Feddersen, and A. A. Scott. 1995. Hantaviruses: clinical, microbiologic, and epidemiologic aspects. Crit. Rev. Clin. Lab. Sci. 32:469-508.
Hooper, J. W., K. I. Kamrud, F. Elgh, D. Custer, and C. S. Schmaljohn. 1999. DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against Seoul virus infection. Virology 255:269-278.
Houdebine, L. M. 2002. Antibody manufacture in transgenic animals and comparisons with other systems. Curr. Opin. Biotechnol. 13:625-629.
Humphreys, D. P., and D. J. Glover. 2001. Therapeutic antibody production technologies: molecules, applications, expression and purification. Curr. Opin. Drug Discov. Dev. 4:172-185.
Koch, J., M. Liang, I. Queitsch, A. A. Kraus, and E. K. Bautz. 2003. Human recombinant neutralizing antibodies against Hantaan virus G2 protein. Virology 308:64-73.
Kolb, A. F., L. Pewe, J. Webster, S. Perlman, C. B. Whitelaw, and S. G. Siddell. 2001. Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis. J. Virol. 75:2803-2809.
Koletzki, D., R. Schirmbeck, A. Lundkvist, H. Meisel, D. H. Kruger, and R. Ulrich. 2001. DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection. J. Biotechnol. 84:73-78.
Liang, M., M. Mahler, J. Koch, Y. Ji, D. Li, C. Schmaljohn, and E. K. Bautz. 2003. Generation of an HFRS patient-derived neutralizing recombinant antibody to Hantaan virus G1 protein and definition of the neutralizing domain. J. Med. Virol. 69:99-107.
Limonta, J., A. Pedraza, A. Rodriguez, F. M. Freyre, A. M. Barral, F. O. Castro, R. Lleonart, C. A. Gracia, J. V. Gavilondo, and J. de la Fuente. 1995. Production of active anti-CD6 mouse/human chimeric antibodies in the milk of transgenic mice. Immunotechnology 1:107-113.
Maes, P., J. Clement, I. Gavrilovskaya, and M. Van Ranst. 2004. Hantaviruses: immunology, treatment, and prevention. Viral Immunol. 17:481-497.
McClain, D. J., P. L. Summers, S. A. Harrison, A. L. Schmaljohn, and C. S. Schmaljohn. 2000. Clinical evaluation of a vaccinia-vectored Hantaan virus vaccine. J. Med. Virol. 60:77-85.
Nakamura, T., R. Yanagihara, C. J. Gibbs, Jr., H. L. Amyx, and D. C. Gajdusek. 1985. Differential susceptibility and resistance of immunocompetent and immunodeficient mice to fatal Hantaan virus infection. Arch. Virol. 86:109-120.
Netski, D., B. H. Thran, and S. C. St. Jeor. 1999. Sin Nombre virus pathogenesis in Peromyscus maniculatus. J. Virol. 73:585-591.
Newton, D. L., D. Pollock, P. DiTullio, Y. Echelard, M. Harvey, B. Wilburn, J. Williams, H. R. Hoogenboom, J. C. Raus, H. M. Meade, and S. M. Rybak. 1999. Antitransferrin receptor antibody-RNase fusion protein expressed in the mammary gland of transgenic mice. J. Immunol. Methods 231:159-167.
Peters, C. J., G. L. Simpson, and H. Levy. 1999. Spectrum of hantavirus infection: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Annu. Rev. Med. 50:531-545.
Schmaljohn, C. S., Y. K. Chu, A. L. Schmaljohn, and J. M. Dalrymple. 1990. Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants. J. Virol. 64:3162-3170.
Schmaljohn, C. S., and J. M. Dalrymple. 1983. Analysis of Hantaan virus RNA: evidence for a new genus of Bunyaviridae. Virology 131:482-491.
Sola, I., J. Castilla, B. Pintado, J. M. Sanchez-Morgado, C. B. Whitelaw, A. J. Clark, and L. Enjuanes. 1998. Transgenic mice secreting coronavirus neutralizing antibodies into the milk. J. Virol. 72:3762-3772.
Song, G. 1999. Epidemiological progresses of hemorrhagic fever with renal syndrome in China. Chin. Med. J. (Engl. Ed.) 112:472-477.
van Kuik-Romeijn, P., N. de Groot, E. Hooijberg, and H. A. de Boer. 2000. Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice. Transgenic Res. 9:155-159.
Yamanishi, K., O. Tanishita, M. Tamura, H. Asada, K. Kondo, M. Takagi, I. Yoshida, T. Konobe, and K. Fukai. 1988. Development of inactivated vaccine against virus causing haemorrhagic fever with renal syndrome. Vaccine 6:278-282.
Yanagihara, R., H. L. Amyx, and D. C. Gajdusek. 1985. Experimental infection with Puumala virus, the etiologic agent of nephropathia epidemica, in bank voles (Clethrionomys glareolus). J. Virol. 55:34-38.(Shuyang Yu, Mifang Liang,)