Microneutralization Test for Influenza A and B and

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1980, p /80/ /07$02.00/0 Microneutralization Test for Influenza A and B and Parainfluenza 1 and 2 Viruses That Uses Continuous Cell Lines and Fresh Serum Enhancement ARTHUR L. FRANK,*,ENNIFER PUCK, BONNIE J. HUGHES, AND THOMAS R. CATE Influenza Research Center, Department of Microbiology, Baylor College of Medicine, Houston, Texas A modified microneutralization test for influenza A and B and parainfluenza 1 and 2 viruses was developed. Use of continuous cell lines (Madin-Darby canine kidney and LLC-MK2), hemagglutination for virus detection, and transfer plates and other microtiter equipment resulted in a highly mechanized technique suitable for titrating large numbers of sera easily and relatively inexpensively. Titers of heat-inactivated human sera were enhanced 0.5 to 3.5 log2 by addition of fresh hamster or human serum to the test. Seroconversion rates and identification of seronegative persons were not changed by fresh serum enhancement, but the magnitude of seroconversion was often increased and the distribution of positive titers was broadened. For influenza A and B, seroconversion rates in the microneutralization test were equivalent to those obtained in rhesus monkey kidney tube neutralization tests. For influenza B, seroconversion rates by microneutralization were almost double those obtained with the hemagglutination inhibition test, but the rates were similar for influenza A/USSR (HlNl). Preexisting microneutralization titers correlated well with resistance to infection with influenza B. Limited experience with parainfluenza 1 and 2 was similar to previously reported findings with the tube neutralization test. Neutralization tests that use tubes of primary monkey cells, bits of allantoic membrane (2, 4), or plaque reduction (10, 20) are available for myxo- and paramyxoviruses. However, these methods are most suitable for testing small numbers of sera. We have found them to be too cumbersome, time-consuming, and expensive for use with large numbers of samples. Wulff et al. reported a microneutralization test based on virus grown in rhesus monkey kidney cells (RMK) and assayed by hemagglutination (21). We modified this method for use with established monolayers of two continuous cell lines that require trypsin to support virus growth (5). In addition, fresh serum was found to enhance the titers of heat-inactivated human serum in this system. The method can be mechanized to a high degree and is suitable for assay of large numbers of sera simultaneously. The present report describes the microneutralization test, compares results with those obtained with other methods, and demonstrates the correlation of microneutralization titers with the response of human volunteers to inoculation with influenza B virus. MATERIALS AND METHODS Equipment. Round-bottom, tissue culture-treated, sterile microtiter plates were used (Linbro Div., Flow Laboratories, Hamden, Conn.) along with transfer 426 plates (1) (Cooke Laboratory Products, Div. of Dynatech Laboratories Inc., Alexandria, Va.). Each transfer plate was rested in a dry, sterile, reusable microtiter plate during loading and diluting. Commercially available microtiter accessories (Rotatiter, Microdrop 1, microdiluters, pipette droppers, all from Dynatech Laboratories) were used for all procedures. In addition, 8- or 12-hole manifold suction devices were made in the Baylor College of Medicine metal shop to facilitate removal of medium from large numbers of plates. Tissue culture. Madin-Darby canine kidney (MDCK) and LLC-MK2 cells were originally obtained from Flow Laboratories, McLean, Va., and were maintained by serial passage in our laboratory. For serological tests, cells were grown to confluence in microtiter plates, using Eagle minimum essential medium supplemented with antibiotics (MEME) plus 5 to 10% fetal calf serum. Before use, the medium was replaced with 0.05 ml of serum-free MEME per well. During the part of the test in which virus production was required, the cells were maintained in protein-free MEME supplemented with 2 to 4,ug of trypsin-tolylsulfonyl phenylalanyl chloromethyl ketone (Worthington Biochemicals Corp., Freehold, N.J.) per ml. MDCK was used for influenza A/USSR and most influenza B/HK tests. LLC-MK2 was used for parainfluenza 1 and 2 and some influenza B/HK tests. Neither cell line gave consistently adequate hemagglutinin production with parainfluenza 3 virus under these conditions. RMK tubes for tube neutralization tests were obtained from Flow Laboratories and maintained as re-

2 VOL. 12, 1980 ported previously (5). Secondary monolayers in microtiter plates made from these tubes were used in some neutralization tests. Titrations and neutralization tests were also performed by the cell suspension method of Wulff et al. (21), using RMK, MDCK, or LLC-MK2 cells. For continuous lines, a high concentration of cells was added to the test initially and a small amount of protein (0.5% fetal calf serum) was included during the 18-h absorption period. In this way an adherent, confluent cell sheet was established before the virus production period, when only trypsin-containing protein, free medium was used. Viruses. The following influenza viruses were used: A/HlNeql (antigenically hybridized from A/USSR/ 92/77 and A/eql/Prague/56) and B/Hong Kong/5/ 72. Each virus was provided by the Center for Disease Control, Atlanta, Ga., and further passaged four to five times in embryonated hen eggs before use in neutralization tests. Parainfluenza virus pools were prepared in RMK, using National Institutes of Health research reference seed pools. Titrations. A virus pool or a dilution selected for use in a neutralization test was decimally diluted in tubes containing protein-free MEME. Then, ml of each dilution per well was dropped onto 4 to 24 wells of a transfer plate previously loaded with ml of MEME per well and transferred to ceil monolayers. Virus titers, in 50% tissue culture infective doses (TCID50), were calculated by the Karber method (9) Ṡera. Sera for use in development and standardization of the microneutralization test were obtained from laboratory personnel, children, and adults participating in a longitudinal study of respiratory virus infections (6) and from young adults participating in several vaccine trials and challenge studies. Some of the latter sera were provided by John Zahradnik. Sera to be titrated were generally inactivated at 56 C for 30 min. However, some longitudinal study sera (natural infection with influenza B/HK/72) were in short supply, had undergone multiple freeze-and-thaw cycles, and were not inactivated further in a few experiments. Pooled animal sera for neutralization enhancement were obtained commercially (Flow Laboratories, and Microbiological Associates, Bethesda, Md.), as gifts from P. Wyde, or directly from hamsters. Hamsters were from a pathogen-free colony maintained by John Trentin (Department of Experimental Biology, Baylor College of Medicine, Houston, Tex.). Sera for enhancement were lyophilized (commercial guinea pig complement) or stored at -70 C and used fresh in each test. HI and tube neutralization tests. For standard hemagglutination inhibition (HI) tests (4) receptor destroying enzyme-treated sera were usually absorbed with chicken erythrocytes, and the test was done starting at a serum dilution of 1:4. However, longitudinal study sera were receptor destroying enzyme treated only, and the tests were done with sera starting at 1:10 dilutions. Tube neutralization tests were done INFLUENZA VIRUS MICRONEUTRALIZATION TEST 427 by using standard methods (4) without fresh serum enhancement. Microneutralization test. Sera were serially diluted in transfer plates loaded with ml of MEME per well, using ml microdiluters. A dilution of virus estimated to contain 20 to 100 TCID50 of virus in ml was prepared in protein-free MEME, and fresh serum for enhancement was added if desired. Virus (0.025 ml/well) was then added to the transfer plates containing test sera, and they were incubated at room temperature (37 C on a few occasions) for 0.5 to 1 h. After incubation, the virus-serum mixtures were transferrred to the monolayer plates, and virus was allowed to adsorb for 18 to 24 h. Ail of the medium and neutralization mixture was then aspirated; the plates were refed with 0.1 ml of protein-free MEME per well, containing 2 to 4 ug of trypsin per ml, and incubated in 5% C02 at 34 C for 5 to 8 days. Then, ml of 1% chicken erythrocytes in phosphatebuffered saline was added per well. After 1 to 4 h at room temperature, absence of macroscropically visible hemagglutination in a well was interpreted as neutralization. At least two microtiter rows were run for each serum sample. Neutralization titers were calculated, using the Karber method (9), as the dilution (in log2) giving 50% neutralization and expressed arithmetically as the reciprocal of the dilution. Performance of the test with a cell suspension was as described by Wulff et al. (21) except for the tissue culture modifications noted above. RESULTS Titrations. Reproducibility of virus titrations by the microtiter method was evaluated by calculating the geometric mean titer (expressed as log1o per ml) and standard deviation for ail titrations of each virus pool on a particular cell type. None of the titers was different from those obtained on the same pools by standard tube titrations. A total of 49 titrations of two pools of influenza A/HlNeql were done on two cell Unes, MDCK and LLC-MK2. The standard deviations of the four means ranged from 0.47 to 0.53 logo TCIDso/0.025 ml. For 31 influenza B/HK titrations the standard deviations were 0.23 and 0.46 logo TCID5w/0.025 ml on MDCK and LLC- MK2, respectively. Eleven titrations of parainfluenza 1 or 2 on LLC-MK2 yielded standard deviations of 0.21 to 0.31 logo TCID50/0.025 ml. Comparison of different test procedures. A series of serum pairs were tested for neutralizing titer, using cell suspensions and established cell sheets of MDCK in the same experiment. Of 28 serum pairs, 14 showed seroconversion to influenza A/USSR (HlNl) or B/HK/72 with established monolayers and 13 showed seroconversion with a cell suspension. Titrations of influenza virus by these two methods gave s2-fold differences in titer. We judged the methods to provide equivalent results, but established monolayers have the advantage of permitting examination before the test is performed in order to assure optimal ceil cultures. Experiments were done comparing neutrali-

3 428 FRANK ET AL. zation of influenza A/HlNeql at 200C (room temperature) or 37 C. Seven sera were tested with fresh serum enhancement, and one serum was tested twice without enhancement. In all instances titers varied s2-fold with the different incubation temperatures. Thus, the remainder of the tests reported were performed with incubation at room temperature. Results of the microneutralization test that used RMK or continuous cell lines were compared. If data from separate tests are pooled, 12 seroconversions were detected with either type of cell among 19 persons naturally infected with influenza B/HK. For seven persons given a live influenza A/USSR (HlNl) vaccine, five seroconverted with MDCK and six seroconverted with RMK. Characteristics of fresh serum enhancement. Testing of the neutralizing titers of fresh human and animal sera with or without inactivation at 560C for 30 min indicated the presence of a neutralizing or enhancing factor(s) in fresh serum. Titers of five lots of fresh hamster serum ranged from 6 to 18 against influenza and parainfluenza viruses; after inactivation, titers feu to <2 or 3. Normal rabbit serum, mouse serum, and two lots of commercial guinea pig serum with fresh serum titers of 11 to 192 against influenza A/USSR (HlN1) had titers of s2 after inactivation. Another lot of guinea pig serum had a decrease in titer from 128 to 32. Nine fresh human sera were tested against influenza A or B, and titers fell 0.5 to 3 log2 when heat inactivated. Because of this evidence of a fresh serum neutralizing factor, a dilution of fresh hamster serum higher than the neutralizing titer of the fresh serum itself was selected to add to the virus pool in the neutralization test. Generally, fresh hamster sera with titers of 6 to 18 were used at dilutions of 1:40 or 1:50 and had no effect on the measured TCID50 of virus in the test. Fresh human serum was used in the same way in a few tests. Seven heat-inactivated human sera were tested (some twice) for enhancement of antiinfluenza A/USSR (HlNl) or B/HK/72 titers with identical dilutions of fresh and heat-inactivated hamster sera. Eleven comparisons showed enhancement with fresh serum only. In one instance, twofold enhancement seemed to occur with one inactivated serum. Table 1 shows the effect of fresh serum enhancement in a series of comparisons of titers of a single serum. Fresh hamster or human serum enhanced the titers of negative or very low titer sera infrequently, enhanced sera with titers of c4 moderately, but enhanced sera with titers of J. CLIN. MICROBIOL. -8 frequently and substantially. The general effect was to spread out the range of titers obtained. Since fresh serum enhancement sometimes increased pre- as well as postinfection titers, the effect on magnitude of seroconversions observed was mixed. Twenty serum pairs were tested one to four times for enhancement of neutralizing antibody titer against influenza A or B. Of 38 comparisons within the same test, 21 showed larger seroconversions, 10 showed smaller seroconversions, and 7 showed no change. It is important to note that, aside from magnitude, unenhanced neutralization was adequate for detection of seroconversion. In the 38 comparisons, enhanced and unenhanced methods each detected the seroconversion 34 times. Three sera were titrated against parainfluenza 1 or 2 virus (or both) with or without dilutions of fresh serum in the same test. In each case enhancement was seen with at least one high dilution of fresh serum. Reproducibility of neutralizing titers. Duplicate samples of 24 sera were coded and tested blindly for fresh serum-enhanced antiinfluenza B/HK titer in the same test on two separate occasions. Thirty sera were tested for anti-influenza A/USSR (HlNi) activity in the same way on one occasion. Ninety percent of the A/USSR (HlNl) serum pairs gave titers that varied s0.5 log2 (1.4-fold) in the same test, 96% varied si log2 (2-fold), and 100% varied -1.5 log2 (2.8-fold). Of the 48 influenza B test pairs, 87.5% varied sl log2 (2-fold) and 96% varied s1.5 log2 (2.8-fold). Thus, it was possible to interpret a fourfold difference between sera on the same microneutralization test as a true difference with 95% confidence. Comparison of the microneutralization method with other serological methods. Table 2 shows results on four groups of sera that were used to compare the ability of HI, tube neutralization, and microneutralization to detect seroconversions. It can be seen that the microneutralization test was an adequate substitute for the standard tube test. Of 74 serum pairs that had both tests performed, 23 pairs showed seroconversions by both tests; 42, by neither test; 4, by microneutralization only; and 5, by tube neutralization only. Most microneutralization tests utiized fresh serum enhancement. There was no evidence of an increased seroconversion rate to influenza A/USSR microneutralization as compared with HI. However, in influenza B infections (Table 2), more seroconversions were detected by microneutralization than by HI. Of all 61 influenza B serum pairs tested by both methods, 26% seroconverted by

4 VOL. 12, 1980 INFLUENZA VIRUS MICRONEUTRALIZATION TEST 429 TABLE 1. Fresh serum enhancement of microneutralization titers No. of Unen- % of comparisons where titer of same serum: Antigen compari- hanced ti- Remained the same or sons' ter decreased rangee) Increased (range) Influenza B/HK 26 '4 31 (0-0.5) 69 ( ) (0-1.5) 82 ( )c Influenza A/USSR (HlNl) 24 c4 79 (0-1.5) 21 ( )d (0) 90 ( ) Either influenza A or influenza B 32 c2 84 (0-0.5) 16 ( ) a Comparisons of titer with and without added fresh serum. Some sera were used on more than one occasion and are listed as separate comparisons. b Range of change in titer in log log2. c 32% of the 73 sera tested increased d Single value of +5.0 excluded from range. TABLE 2. Comparison of seroconversions detected by HI, tube neutralization, and microneutralization Influenza virus exposure No. of seroconversions detected by: No. of serum pairs with indi- Intranasal Natural in- Id catea tests" inyeocula- Tub ne- Mcrnu tionb fectionc tralization tralization' 42 B/HK/ B/HK/ f A/USSR (HlNl) A/USSR (HlNl) a The groups were selected for comparison of tests. b Inoculation of volunteers; not all volunteers infected. c Culture-proven infections only in 15. d For the second group, HI test was done starting at a serum dilution of 1:10; for the other two groups the starting serum dilution was 1:4. e All fresh serum enhanced except for 11 of 14 in second group. f-, Test not performed on sera in this group. HI and 46% seroconverted by microneutralization. Results were similar if only the 38 pairs among volunteers who resisted infection (Wil- neutralization tests were significantly higher associated with virus excretion were compared; 26% seroconverted by HI and 56% seroconverted by microneutralization. The microneutralization test with enhancement was compared with other serological methods for accuracy in predicting susceptibility of volunteers to infection after intranasal inoculation with an influenza B virus (Table 3). The volunteers were selected so that most had little or no detectable HI antibody, and any antibody present was derived from remote natural infection. Two different viruses having surface antigens closely related to influenza B/HK/72 were used as inocula: a cold-adapted recombinant live virus vaccine candidate and a wild-type challenge pool prepared from a 1977 isolate. (A complete description of these studies will be presented elsewhere [T. R. Cate and R. B. Couch, manuscript in preparation].) Although these two inocula differ in the illnesses they produce, their infectivities for persons with differing levels of preinoculation antibody were very similar, and results were combined for Table 3. Preinoculation titers measured by HI or tube coxon, P < 0.05, two-tailed in each case). However, among the large proportion of volunteers with very low titers in these tests (26 of 32 with titers of c4 by HI; 21 of 32 with titers of c2 by tube neutralization), several failed to become infected. Preinoculation titers measured by microneutralization were more broadly distributed, and resistance to infection by those with the higher titers was highly significant (Wilcoxon, P < 0.01, two-tailed). Most importantly, at least some antibody was detected in the microneutralization test that used fresh serum enhancement in all volunteers who subsequently failed to shed virus or resisted infection completely. Ail of the 11 volunteers with little or no antibody (c2) detectable in this test did become infected. Parainfluenza 1 and 2 microneutralization. The microneutralization procedure developed for influenza viruses was adapted for use with parainfluenza viruses 1 and 2 after confirming the reliability of titrations and the existence of fresh serum enhancement (noted previously). LLC-MK2 cells were used. Results from 17 infected persons are presented in Table 4. The

5 430 FRANK ET AL. J. CLIN. MICROBIOL. TABLE 3. Correlation ofpreinoculation antibody titers with evidence of infection after intranasal inoculation of volunteers with influenza B viruses' Preinoculation antibody No. of volunteers with: Test system Titer No. of volun- Virus Seroconteers sheddig versioninfectiond HI < (100) (65) (50) (0) Tube neutralization < il 14 (88) (40) (57) (33) (0) Microneutralizatione < (100) 2 2 O (57) 4 2 O i 2 (58) O i ( O i O 0 O O (0) a Viruses were a live virus vaccine candidate and a wild-type virus challenge pool. b Virus recovered from nasal wash specimens. C Fourfold or greater rise in titer obtained with any test. Of the 16 seroconversions, 6 were by all three tests, 8 were by the two neutralization tests without HI, 1 was by microneutralization and HI, and 1 was by tube neutralization alone, although the latter volunteer also shed virus. d Virus shedding or seroconversion, or both. e Fresh serum enhancement used. Downloaded from TABLE 4. Microneutralization test results in persons infected with parainfluenza virus types 1 and 2 Microneutralization test result- Parainfluenza Age No of sevirus infec- group N Parainflu- Parainflutiofla (Mo) rum pairs enza i enza 2 serocon- seroconversion version Type 1 ' > Adults Type 2 > oc 3 a Natural infection, culture proven, in persons participating in the longitudinal study. b Fresh serum enhancement used. 'One pair not tested for heterologous seroconversion. neutralization test demonstrated a serological rise in 59% of these people, and there were two heterologous seroconversions. DISCUSSION The neutralization technique described in this report consists of adaptations of previously available methods and principles. The main components are (i) continuous cell lines dependent on trypsin for myxo- and paramyxovirus replication (5), (ii) detection of virus production macroscopically by hemagglutination (21), (iii) enhancement of neutralization titers with fresh serum, and (iv) commercially available microtiter equipment. This technique offers advantages over previously published methods in terms of time, materials, and cost when titrating large numbers of sera. The cell lines used are readily available and relatively inexpensive. The test can be highly mechanized and easily interpreted. Further mechanization is possible (13). Results are at least as good as those obtained with the standard tube neutralization test. One element of the test, fresh serum enhancement, has apparently not been applied previously in human serological studies with influenza or parainfluenza viruses. The existence of such enhancement for these viruses has been previously documented, however. Enhancement of HI titers by complement was demonstrated for influenza A virus (16), and complement enhancement of neutralization was demonstrated for on September 24, 2018 by guest

6 VOL. 12, 1980 Newcastle disease virus (11). Extensive work was also done on a thermolabile noncomplement cofactor, thought to be a lipoprotein, that enhanced neutralization of influenza A and Sendai viruses (18, 19). Complement has been used in neutralization tests for a related virus, respiratory syncytial virus (7, 14), and a different type ofneutralization enhancement, produced by heterologous anti-immunoglobulins, has been utilized with mumps virus (17). Recent reviews summarize previous work characterizing neutralization enhancing factors and the mechanisms by which they operate (3, 12). It is clear that the interactions may be complex and vary in different situations. In this report we have simply described the enhancement factor as labile at 560C for 30 min. Considerable work will be required to identify the mechanism of fresh serum enhancement in this system. We presently see several potential advantages of enhancement. The magnitude of seroconversion is increased in the majority of instances, thus decreasing statistical and technical concerns about interpretation of low-level seroconversions. The distribution of titers is broadened so that populations may be separated more readily by titer level for correlations with other parameters; a suggestion of this was seen in the influenza B vaccinees shown in Table 3. Neutralizing antibody susceptible to enhancement is a component of the immune response not previously studied in clinical settings; it is possible that improved correlations between serological and clinical responses may be found with this technique. On the other hand, our observations did not indicate that enhanced neutralization is necessary for adequate detection of seroconversion; testing without enhancement is probably sufficient for this purpose. Nor did we find evidence that antibody will be found in individuals with unenhanced neutralization titers of c2. The utility of any neutralization test as an alternative or supplement to the HI test for influenza viruses probably varies with the population studied and the question asked. One example is a recent vaccine trial reported by Gross and Davis (8). They found the preimmunization level of neutralizing antibody to be more useful than HI antibody in predicting response to a recombinant influenza A (HswNl) vaccine. INFLUENZA VIRUS MICRONEUTRALIZATION TEST 431 Kilbourne (10) noted a dissociation of HI and neutralizing antibody in a vaccine trial in which heterotypic HI rises were associated with little neutralizing activity. In a study of influenza A/ Victoria (H3N2) live virus vaccines, the neutralization test gave a higher number of seroconversions than did HI (Cate and Couch, in preparation). A correlation of cord serum anti-influenza A (H3N2) titer and occurrence of influenza infection in infants was recently obtained in our laboratory with the microneutralization test (15). In this situation, the ability to test for low levels of antibody was important, as it was for Gross and Davis (8). Our data indicate that for influenza B/HK the seroconversion rate was almost doubled by the neutralization test as compared with HI. Wright et al. (20) reported even better results with a plaque reduction test. However, seroconversion rates for influenza A/ USSR (HlNl) by neutralization and HI were not different in the populations we have studied so far. The value of the neutralization test for study of parainfluenza 1 and 2 virus infection was discussed briefly by Chanock (2). The main problems are frequent cross-reactions and sensitivity that is only moderate. Results with our small group of sera (Table 4) seem to demonstrate both of these problems. ACKNOWLEDGMENTS We are grateful to W. P. Glezen for important early suggestions and to R. B. Couch for suggestions, help with some experiments, and review of the manuscript. This study was supported by Public Health Service contracts AI and AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Catalano, L. W., D. A. Fuccillo, and J. L. Sever Piggy-back microtransfer technique. Appl. Microbiol. 18: Chanock, R. M Parainfluenza viruses, p In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial and chlamydial infections. American Public Health Association, Washington, D.C. 3. Cooper, N. R., and R. M. Welsh, Jr Antibody and complement-dependent viral neutralization. Springer Semin. Immunopathol. 2: Dowdle, W. A., A. P. Kendal, and G. R. Noble Influenza, p In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial and chlamydial infections. American Public Health Association, Washington, D.C. 5. Frank, A. L., R. B. Couch, C. Griffis, and B. Baxter Comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses. J. Clin. 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7 432 FRANK ET AL. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial and chlamydial infections. American Public Health Association, Washington, D.C. 10. Kilbourne, E. D Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in induction of antibody to neuraminidase in humans. J. Infect. Dis. 134: Linscott, W. D., and W. E. Levinson Complement components required for virus neutralization by early immunoglobulin antibod;l. Proc. Natl. Acad. Sci. U.S.A. 64: Mandel, B Neutralization of animal viruses. Adv. Virus Res. 23: Mayner, R. E., D. J. MeDorman, B. C. Meyer, and P. D. Parkman Automated microtransfer technique for the assay of poliovirus and mumps virus neutralizing antibodies. Appl. Microbiol. 28: Mills, J., J. E. Van Kirk, P. F. Wright, and R. M. Chanock Experimental respiratory syncytial virus infection of adults. J. Immunol. 107: Puck, J., W. Glezen, A. Frank, and H. Six Protection of infants from influenza A infection by transplacentally acquired antibody, p In J. J. CLIN. MICROBIOL. D. Nelson and C. Grassi (ed.), Current chemotherapy and infectious disease, vol. II. American Society for Microbiology, Washington, D.C. 16. Reno, P. W., and E. M. Hoffman Enhancement of hemagglutination inhibition by complement. Infect. Immun. 6: Sato, H., P. Albrecht, J. T. Hicks, B. C. Meyer, and F. A. Ennis Sensitive neutralization test for virus antibody. 1. Mumps antibody. Arch. Virol. 58: Smorodintsev, A. A., and A. A. Yabrov The mechanism of enhanced activity of anti-influenza virus neutralizing antisera on their interaction with native serum from normal animals. Acta Virol. 7: Styk, B Cofactor and specific antibodies against influenza viruses. XI. Mechanism of the action of antibody cofactor. Acta Virol. 9: * 20. WrightP. F., J. D. Bryant, and D. T. Karzon A comparison of influenza B/HK infections in infants, children and young adults. J. Infect. Dis. 141: Wulff, H., J. Sveken, J. D. Poland, and T. D. Y. Chin A new microneutralization test for antibody determination and typing of parainfluenza and influenza viruses. Proc. Soc. Exp. Biol. Med. 125: Downloaded from on September 24, 2018 by guest