Human Immunoglobulin Specificity After Group B Arbovirus Infections

Transcription

1 INFECTION AND IMMUNITY, Sept. 97, p American Society for Microbiology Vol. 6, No. 3 Printed in U.S.A. Human Immunoglobulin Specificity After Group B Arbovirus Infections ROBERT McNAIR SCOTT, JACK M. McCOWN, AND PHILIP K. RUSSELL Departmentt of Virus Diseases, Walter Reed Army Itistitute of Research, Washinigtoni, D.C. Received for publication 6 April 97 Sera obtained during a dengue- epidemic from individuals with primary or secondary group B arbovirus infections contained cross-reactive antibody against several viruses in this group. Hemagglutination-inhibition antibody titers of the immunoglobulin M fractions from these sera revealed a rise in antibody specific for dengue-. The acute and convalescent sera in over half of the secondary infection group also had low-level unchanging immunoglobulin M antibody titers to yellow fever which implicated yellow fever immunization as the primary infection. Type-specific serological diagnosis of group B arbovirus infections is sometimes possible in primary cases; however, precise identification of the infecting virus by usual serological means in subsequent infections with group B arboviruses is precluded by the development of antibody that exhibits extensive cross-reactions among members of this group. A number of studies in animals with closely related viral agents have demonstrated greater specificity of immunoglobulin M (IgM) for the homologous virus as compared to immunoglobulin G (IgG;, -6,, 7). Studies with group B arboviruses in rabbits (7) have suggested that the specificity of IgM might be applied to the serological diagnosis of human arbovirus disease. Our studies were designed to determine whether the viral specificity of IgM antibody in sera collected from patients during a dengue- epidemic is different from whole sera and, if so, whether this specificity could be useful as a diagnostic tool. MATERIALS AND METHODS Sera. Blood was collected from military personnel or their dependents presenting with clinically diagnosed dengue fever at three military clinics in San Juan, Puerto Rico, during the summer of 969. All patients had a benign self-limiting disease with no sequelae. All military personnel but not their dependents had previously been immunized with 7-D yellow fever virus vaccine. Sera were separated and stored at - C. Separation and identification of immunoglobulins. Serum samples were absorbed with goose red blood cells, and.3 ml was applied to a.-ml to % linear sucrose gradient. Gradients were centrifuged in a Beckman SW39 rotor at, X g for hr;.3-ml fractions were collected through the bottom of the gradient tube. Single radial diffusion on commercially obtained agar plates (Hyland Laboratories) was used for identification and quantification of immunoglobulins. Sucrose fractions containing IgM were pooled and tested for antibody activity. HI test. Hemagglutination-inhibition (HI) tests were done by a microtiter modification of the method of Clarke and Casals (3). Whole sera were extracted with acetone before testing. Antigens made from prototype strains () of yellow fever (YF), Japanese encephalitis (JE), and the four dengue viruses were diluted with borate saline buffer to contain eight hemagglutinating units at their optimum ph. Sensitivity of HI antibody activity to reduction by -mercaptoethanol was tested by incubating. ml of each antibody sample with an equal volume of. M -mercaptoethanol in borate saline (ph 9.) for hr at room temperature, followed by standard dilution and testing for HI. Plaque-reduction neutralization test. Virus-neutralizing activity of whole sera and IgM fractions were measured by a plaque reduction neutralization test (). Viruses used were dengue-l (Hawaii), dengue-3 (H-7), dengue- (PR-9), dengue- (375), and yellow fever (French Neurotropic). Sensitivity of neutralizing antibodies to reduction was determined by incubating antibody dilutions for 3 hr with an equal volume of.3 M ethanethiol, and then heating the nmixture at 56 C for 3 min to remove the volatile mercapton before testing (). Virus isolation and identification. Virus isolation was carried out in LLC-MK cell cultures by methods previously described (). Viruses were identified by using a plaque-reduction neutralization test () with hyperimmune ascitic fluids prepared against dengue virus in mice. RESULTS Fourteen patients with significant increases in HI antibody against dengue- antigen were selected for study. Patients were divided into two groups on the basis of the HI antibody response 77 Downloaded from on July 7, by guest

2 7 SCOTT, McCOWN, AND RUSSELL INFECT. IMMULNITY to group B arboviruses. The first group consisted of four patients with low ( < : ) convalescent HI antibody titers and minimal cross-reactions with JE virus. These patients were presumed to have had a primary reaction to their first group B arbovirus infection (Table ). Dengue- virus was isolated from two of these patients. All four were dependents of military personnel. The second group consisted of patients (all military personnel) with high level (>:6) HI antibody responses and marked cross-reactions among the group B antigens (Table ). In this group, the recent dengue infection was considered to be secondary to a previous group B experience, resulting in an anamnestic response. Dengue- virus was isolated from five acute sera in this group. The acute sera of half of the second group contained HI antibodies against YF but not dengue or JE viruses. Since specific low-level HI titers have been demonstrated to persist in persons immunized with YF (9) and since military personnel had undergone YF immunization, this probably represented persistent YF antibody. The remain- TABLE. Hemagglutinlationi-inihibitioni (HI) a,itibody titers in whole sera from patienits with primary groutp B arbovirus intfectionis with denigue- Patient no. Virus recovered Day of disease DEN- DEN- DEN-3 DEN- YF JE PR- None 3 <' < < < < 3 < < PR- DEN- 3 < < < < < < 5 < PR- None < < < < < < + I < < PR-5 DEN- < < < < < < < < < DEN, Dengue; YF, yellow fever; JE, Japanese encephalitis. Reciprocal HI titer against units of antigen. TABLE. Heniagglutintationt-inihibitioii (HI) a,itibody liters in whole sera fromii patients wit/h seconialciry grouip B inifectioni wit/i dengue- a Patient no. V-irus recovered Day of disease DEN- DEN- DEN-3 DJEN- YF JE PR- None I <6 < < < < 3 3,56, 3 3 PR- None 5,,,,56,, PR-5 None ,,56, PR-33 DEN-,, 5,,56,56, PR-39 DEN- < < < < < 6,,56,56 5,,56 PR- DEN-? < < < < < < +,, 5,,56 5, 6 PR-7 None 6,,56,56,56 5,,56,56 5, 5,,56 5,,56 PR- None < < < + 6,,56, 5,, PR-56 DEN- < < < < 3,56 6,56 PR-6 DEN ,,56,,,, DEN, Dengue; YF, yellow fever; JE, Japanese encephalitis. Reciprocal HI titer against units of antigen. Downloaded from on July 7, by guest

3 VOL. 6, 97 IGM ANTIBODY AFTER ARBOVIRUS INFECTION 79 ing acute sera had heterospecific antibodies to all HI antigens tested. These individuals may have had a previous group B arbovirus infection after the YF immunization, but prior to the dengue- infection, or, more likely, the heterospecific antibody might be accounted for by collection of the acute sera after the onset of the anamnestic antibody production. When convalescent sera from the primary group was treated with -mercaptoethanol, there was a fourfold decrease in HI titers, whereas -mercaptoethanol had no effect on the convalescent HI titers in the second group. Separation of serum immunoglobulins was carried out in sucrose gradients; IgM was usually found in the lower four fractions and IgG above fraction four. Fractions to 3 from each gradient were pooled and assayed for IgM and IgG content. A relatively constant proportion of IgM, ranging between 9 and of the whole serum concentration, was extracted by this means; IgG concentrations in the IgM fractions were less than 5 %f mg. HI antibody activity of the IgM fractions showed a rise in titer against the homotypic dengue- antigen in all individuals. In the four primary cases, dengue- was the only antigen recognized by the IgM fractions (Table 3). In the convalescent sera from secondary cases, there was a similar lack of cross-reactivity of IgM with heterologous dengue and JE antigens. The magnitude of the dengue- IgM response was similar in the two groups (Table ). However, in 6 out of of the patients in the secondary group, there was also IgM antibody activity against the YF antigen. In these cases, anti-yf activity was found in both the acute and convalescent serum. Plaque-reduction neutralization studies were performed on convalescent sera from three individuals with secondary antibody responses. Whole convalescent serum was found to neutralize all four denaue seritypes and YF (Table 5). The IgM fractions obtained from these convalescent sera again showed specific activity against dengue-, with little or no cross-reactivity against the other dengue types. As expected from the HI data, YFneutralizing activity was found in the IgM fractions of the two sera tested. DISCUSSION The most important practical aspect of these observations is the fact that the specificity of the IgM antibody can be used to make a type-specific serological diagnosis in secondary group B arbovirus infections. Herein we have demonstrated the specificity of the IgM response in dengue- infections in persons immunized against yellow fever. A similar observation was made in JE infections after dengue infections in both man and gibbons (R. Edelman, personal communication). The usefulness is limited, however, to situations where the infecting group B agents have distinct antigenic differences, since little or no IgM response occurs when both infections are due to very closely related agents, such as within the dengue virus group (, 3). The reasons for a higher degree of specificity for IgM antibody compared to IgG are as yet unclear. Differences in combining sites on the antigen for IgM and IgG antibodies may play a role. Studies of IgM and IgG antibodies against foot-andmouth disease virus have suggested a difference in the size of the antibody-combining sites for the two immunoglobulins (). Electron micrographs revealed that IgG reacted with sites over the entire surface of the virion, and IgM reacted with only one site at regularly spaced intervals on the viral particle (). Although specific IgM has been described in several animal systems after primary immunization (-6, ), this does not appear to be universally true (, 5); our da.ta suggest that TABLE 3. Hemagglutitnationi-ilihibitionz (HI) alttibody liters of serum immunloglobutliln M (gm) fractionis from patielnts wit/i primary grouip B arbovirus inlfretionis wit/i denigute- Downloaded from on July 7, by guest Patient no. Day of disease IgMI fraction/ whole serum DEN- DEN- DEN-3 DEN- YF JE PR- 3.3 NT NT NT NT NT 3.3 ') PR NT NT NT NT NT.39 3 PR-.3 NT NT NT NT NT +.33 PR-5.3 NT NT NT NT NT.36 a DEN, Dengue; YF, yellow fever; JE, Japanese encephalitis; NT, not tested;, no activity undiluted. b Reciprocal HI titer against units of antigen.

4 SCOTT, McCOWN, AND RUSSELL INFECT. IMMIJNITY TABLE. Hemagglutiniationi-inihibitiont (HI) anitibody titers of serum immunzoglobulini M (IgM) fractionis from patienits with seconidary group B arbovirus inifectionis with denigue- Patient no. PR- PR- PR-5 PR-33 PR-39 PR- PR-7 PR- PR-56 PR-6 Day of disease I- I gm% fraction/ wvhole serum DEN- DEN- b DEN-3 Antigena DEN- a DEN, Dengue; YF, yellow fever; JE, Japanese encephalitis;, no activity undiluted. b;reciprocal HI titer against units of antigen. TABLE 5. Neutralization tests on whole convalescenit sera anzd immunioglobulin M (IgM) fractions Viruses Patient no. DENa- DEN- DEN-3 DEN- YF Whole IgMb Whole IgM Whole IgM Whole IgM Whole IgM serum fractions serum fractions serum fractions serum fractions serum fractions PR-39 6c < 7 3 < < 7 < 56 PR- 6 < 5 5 < 5 < 6 PR-7 3 < 5 > < a DEN, Dengue. b IgM concentration adjusted to that of whole serum. C Reciprocal of 5%v plaque-reduction titer. humans have the capability of producing specific IgM after both primary and secondary experiences with group B arboviruses. The antibody response to a secondary infection with a group B arbovirus appears to be a mixture of an anamnestic response to some antigenic determinants which are very similar or identical to those of the first virus, and a primary response to other determinants which are unique to the second virus. In the cases reported here, common group B antigens to which the host had been exposed through prior YF immunization lead to an anamnestic response when the dengue infection occurred, whereas dengue type-specific antigenic determinants not previously seen produced primary responses with IgM antibody. Thus, the specificity of IgM may be due in part to exclusion. The observation of unchanging titers of IgM antibody directed against YF in 6 of patients showing secondary HI titers is consistent with the report that IgM antibody has been found to persist for as long as months after YF immunization. It is noteworthy that, in spite of major rises in titers of cross-reacting IgG antibody and a rise in anti-dengue IgM antibody, the anti-yf IgM antibody remained constant, indicating that completely separate antigenic determinants are involved. YF JE Downloaded from on July 7, by guest

5 VOL. 6, 97 IGM ANTIBODY AFTER ARBOVIRUS INFECTION ACKNOWLEDGMENTS We thank Walter E. Brandt and Earl H. Fife, Jr., for their help and encouragemenit in the preparation of this manuscript, and M. Frances Byrd for her clerical assistance; LITERATURE CITED. Brown, F., and C. J Smale. 97. Demonstration of three specific sites on the surface of foot-and-mouth disease virus by antibody complexing. J. Gen. Virol. 7:5-7.. Catalogue of Arthropod-Borne Viruses of the World Public Health Service Publication no. 76. U.S. Government Printing Office, Washington, D.C. 3. Clarke, D. H., and J. Casals. 95. Techniques for hemagglutination and hemagglutination inhibition with arthropodborne viruses. Amer. J. Trop. Med. Hyg. 7: Cowan, J. M. 97. Immunechemical studies of foot-andmouth disease. VI. Differences in antigenic determinant site recognition by guinea pig 9S and 7S antibodies. J. Immunol. : Hampar, B., L. M. Martos, D. V. Ablashi, R. F. Siguenza, and G. A. Wells Differentiation of cross-reacting simian cytomegalovirus strains by late 9S rabbit neutralizing antibodies. J. Immunol. 3: Hampar, B., L. M. Martos, M. Chakrabarty, and M. A. K. Burroughs. 97. Late 9S rabbit antibody neutralization test for differentiating herpes simplex virus types and. J. Immunol. : Monath, T. P. C. 97. Neutralizing antibody responses in the major immunoglobulin classes to yellow fever 7D vaccination of humans. Amer. J. Epidemiol. 93:-9.. Murray, E. S., J. M. O'Connor, and J. A. Gaon Differentiation of 9S and 7S complement fixing antibodies in primary versus recrudescent typhus by either ethanethiol or heat. Proc. Soc. Exper. Biol. Med. 9: Pond, W. L., N. J. Ehrenkranz, J. X. Danauskas, and M. J. Carter Heterotypic serologic responses after yellow fever vaccination detection of persons with past St. Louis encephalitis or dengue. J. Immunol. 9: Russell, P. K., A. Intavivat, and S. Kanchanapilant Anti-dengue immunoglobulins and serum Blc/a globulin levels in dengue shock syndrome. J. Immunol. :-.. Russell, P. K., and N. Nisalak Dengue virus identification by the plaque reduction neutralization test. J. Immunol. 99: Russell, P. K., A. Nisalak, P. Sukhavachana, and S. Vivona A plaque reduction test for dengue virus neutralizing antibodies. J. Immunol. 99: Russell, P. K., S. Udomsakdi, and S. B. Halstead Antibody response in dengue and dengue hemorrhagic fever. Jap. J. Med. Sci. Biol. (Suppl.) :3-.. Wagner, G. G., and K. M. Cowan. 97. Immunochemical studies of foot-and-mouth disease. IX. Differences in neutralizing activities of guinea pig and bovine 9S and 7S antibodies. J. Immunol. : Webster, R. G. 96. The immune response to influenza virus. III. Changes in the avidity and specificity of early IgM and IgG antibodies. Immunology : Westaway, E. G. 96. Antibody responses in rabbits to group B arbovirus kunjin: serologic activity of the fractionated immunoglobulins in homologus and heterologous reactions. J. Immunol. : Westaway, E. G. 96. Greater specificity of 9S and 7S antibodies on haemagglutination-inhibition tests with closely related group B arboviruses. Nature (London) 9: Yuill, T. M., P. Sukhavachana, A. Nisalak, and P. K. Russell. 96. Dengue-virus recovery by direct and delayed plaques in LLC-MK cells. Amer. J. Trop. Med. 7:-. Downloaded from on July 7, by guest