General discussion—Session III

General Discussion-Session III DR C. B. REIMER. Four of the 6 American manu- facturers producing influenza vaccine this year claim in their package literature that their vaccine has been purified, but no manufacturer claims that his product is pure. The word " purity ", denoting a quality or state of being free from all heterogeneous or extraneous matter, has a ring of absoluteness to it. In contrast, the word " purification " can imply that some process has been performed for refining the material to a relatively cleaner condition than it was in previously. But what is really needed is some operational definition, available to both the manufac- turer and the regulatory authority, which makes a quantitative statement about the relative purity of the product itself, independent of the method used for its purification. For example, with influenza virus vaccine one can define relative purity as a ratio between the amount of specific antigen desirable (such as CCA units per dose) and the amount of material that must be injected (mg of Lowry protein per dose). Thus, the " purity " of influenza vaccines of the 1940s, purified by the then new Sharples cen- trifuge process, contained approximately 2000 CCA units per mg of protein. In comparison with this, some recent experimental vaccines have contained as many as 25 000 CCA units. per mg of protein. Now that we have these cleaner vaccines we know that the older vaccines were less than 10% pure, that is they had 90% impurity. The previous papers have described the problems of standardization of influenza vaccine in the labora- tory. Later speakers will discuss the even greater problems encountered in assessing the safety and effectiveness of vaccine in man. When one considers the problems of obtaining precision with biological measurements, it is not surprising that we do not have an acceptable standard of purity for influenza vaccine. However, there may be a solution. I think that we should examine vaccines directly for the presence of undesirable agents, such as the impurities which do harm. These can be divided conveniently into 3 categories: the host tissue, bacterial contami- nants, and viral contaminants (including rickettsiae and mycoplasmas). To avoid problems of allergic sensitization against egg protein or of eliciting immediate or delayed hypersensitivity in a previously sensitized host, residual egg protein should be absent from modern vaccines. According to Untracht: v a Untracht, S. (1958) N. Y. St. J. Med., 58, 1724. " The incidence of individuals in whom egg sensitivity may be of an order high enough to present a serious hazard frominjection ofchick-egg-propagated vaccine is estimated to be about one to two per 1000 treated persons". Also, human blood group A antigens are present in egg protein and sensitization to these is undesirable, particularly for a potential mother. In anticipation of this Conference, I homogenized 4 11-day-old embryonated hens' eggs, shells and all, and immunized 2 rabbits with the clarified material. By immunoelectrophoresis this rabbit antiserum showed antibody to 11 distinct proteins of my egg antigen. Two weeks ago, I procured in Atlanta a single vial of 1969-70 commercial influenza vaccine of each of the 6 American manufacturers of this product. Furthermore, I procured samples of commercial influenza vaccines from the last few years. Using my rabbit antiserum, it was easy to establish, by double diffusion in agar, that some of these vaccines contained a number of egg antigens. Where precipitation lines were found, the heaviest line formed a clear linkage with recrystallized ovalbumin. The accompanying figure shows a 2-in x 3-in RADIAL DIFFUSION TESTING OF COMMERCIAL INFLUENZA VACCINES A NA microscope slide to demonstrate how these commer- cial vaccines behaved in radial diffusion against a 1/160 dilution of the rabbit antiserum and the area of the precipitation disc is proportional to the amount of egg antigen in each vaccine. The 6 current vaccines are in wells above the letters designating specific manufacturers. Three of these firms have employed recent new processes for purification, and vaccines 2405 - 571- GENERAL DISCUSSION-SESSION III from the same manufacturer occur in the same box. A sample containing 8.0 -g per ml ovalbumin was placed in the lower right well. Current (1969-70) vaccines from manufacturers B and F contain the most soluble egg protein (each 6.5 ,ug per dose). This is much less than the 50 ,tg per dose for the 1966 vaccine from manufacturer A shown in the lower left corner. The current vaccine F was made by the Sharples process, but surprisingly, the vaccine B was not as clean as previous vaccines from this manufac- turer and was made by employing a process suffi- ciently different to require comment in the package literature. Soluble egg protein in the remaining current vaccine ranged from undetectable levels to 2 jug per dose. It must be emphasized that this method does not measure aggregate or particulate material too large to diffuse through the agar. We shall determine this by immunizing mice with the vaccines and measure the development of antibody to egg by radial diffusion, using antigen in the agar. We have found this system most useful for similar studies. These tests are quite economical; 1 ml of rabbit antiserum permits 500 to 1000 analyses, each requiring only 10 pl of vaccine. When processing many hundreds of thousands of eggs per week, it is extraordinarily difficult to keep an occasional heavily contaminated egg out of the vaccine. If only 1 egg per 1000, with a bacterial count of 101, slips through, the bacterial count in the pool is 106. These organisms are killed shortly thereafter by the preservative and the formol, but the bacterial products remain in the vaccine unless removed by subsequent processing. At the National Communicable Disease Center, the Bacterial Chemis- try Unit has made an anti-Salmonella reagent covering all groups A-H. I used this reagent to test these same influenza vaccines by radial diffusion. Fortunately, Salmonella antigens were absent, but traces were found in some experimental influenza vaccines I had made some years ago. Because current influenza vaccines are now cleaner than they were previously and give fewer local and systemic reactions clinically, perhaps pyrogen testing might be the easiest method for assessing and con- trolling bacterial toxins. Influenza vaccine is made from eggs from flocks which are not free from leucosis virus, as discussed by Dr Perkins. This is not considered to be a prob- lem for this killed preparation, but, of course, the situation would be entirely different for a live in- fluenza virus vaccine. Influenza vaccines of the future may require more viral antigen, or may be incorporated in safe adju- vant, or perhaps may be given as aerosols by the respiratory route. Each of these changes will neces- sitate greater attention to the question of relative purity of the immunizing agent. DR T. O'BRIEN. Previous investigators have reported that formol-inactivated influenza virus vac- cines were relatively stable for long periods when stored at 4°C. It was observed that freezing and storage in the frozen state, however, caused the haemagglutinating activity of such vaccines to decrease, and lyophilization resulted in a loss of both the haemagglutinating and immunizing pro- perties. A recent incident, in which the chicken-cell agglutinating (CCA) activity of a zonal-purified formolized influenza vaccine was unaffected when a lot had been accidentally frozen, prompted us to reinvestigate the effects of freezing and lyophilization upon the stability of influenza vaccines prepared either by the conventional Sharples process or by the newer zonal-ultracentrifugation or ether-degrad- ation process. Aliquots of each preparation tested were either held at 4°C for 24 hours, or frozen in a - 20°C freezer or quick-frozen to - 70°C in an ethanol-dry- ice bath. Frozen samples were rapidly thawed under tap water at room temperature immediately before measuring the CCA activity. In our initial experiments, there was evidence that freezing reduced the CCA activity, especially with those inactivated vaccines prepared by the Sharples and ether-degradation processes and which were frozen at - 20°C. Experiments to determine the effects of freezing and long-term storage at 4°C, - 20°C and - 70°C on CCA activity are currently being performed. Pre- liminary results indicate that with these prepara- tions the CCA activity was unaffected by freezing and immediate thawing. However, the CCA activity of Sharples and HA vaccines was considerably reduced after storage at -20°C for 1 week. Al- though the zonal vaccines appear unaffected by storage at - 20°C for 1 week, after 4 weeks the CCA activity had decreased slightly. The CCA contents of Sharples and HA vaccines stored at - 70°C have decreased approximately by 40% after 1 week and 50% after 4 weeks of storage. The zonal vaccines consistently showed increased CCA content as com- pared with the 4°C control at zero time. 572 GENERAL DISCUSSION-SESSION III We have also studied the effect of lyophilization upon the CCA activity of formol-inactivated in- fluenza vaccines and we find that lyophilization causes a significant loss of CCA activity, particularly with the Sharples and HA vaccines. DR J. W. MILLAR. As Dr O'Brien mentioned, some A2/Aichi/2/68 Hong Kong vaccine purified by zonal centrifugation inadvertently became frozen at -70°C during transport. How long it remained frozen is not known, but it was probably 48- 72 hours. On checking the CCA potency, it was found that there had not been any appreciable loss. This vaccine was used in a school study as the second dose of 0.5 ml and for primary immunization in 3 nursing homes in Montgomery County, Mary- land. Excellent rises in HI titres were obtained in both these groups. Twelve medical students were also given 0.5 ml of this frozen vaccine and all but 2 had HI antibody rises after 3 weeks. DR E. B. SELIGMANN, JR. The pyrogen test specified by the US Public Health Regulations for Biological Products is performed in rabbits with the product injected intravenously. A dose of 3 ml per kg of rabbit body-weight is given for most products. The product fails to meet test requirements if one- half or more of all rabbits show a temperature rise of 0.6°C or more or if the average temperature rise of all rabbits is 0.5°C or more. Viral vaccines are included among a number of products specifically exempted from this requirement. Some years ago we experimented with a modification of the pyrogen test with influenza virus vaccine. We arbitrarily used a dose of 0.1 ml per kg of body-weight and found that some vaccines were quite reactive at this dose while others were virtually nonreactive. All vaccines tested at that time were concentrated by Sharples centrifugation and we were unable to develop a correlation between low pyrogenicity and method of manufacture. The presence of killed bacteria in the vaccines complicated the problem. With the development of zonal centrifugation and other methods for purifying influenza virus vaccines, the possibility of using a pyrogen test to evaluate the potential reactivity of a vaccine exists. For this test, 3 rabbits are used in the weight range of 1.7 kg to 3 kg and pre-injection temperatures are taken within 1 hour prior to injection of the product. The vaccine is injected intravenously into the marginal ear vein of the rabbit and thereafter temperatures are taken at hourly intervals for the first 3 hours, the temperature rise at each time interval for each rabbit being recorded. Except for the volume of the dose injected, this pyrogen test meets the requirements of the test specified by the US Public Health Service Regulations. The results of a typical pyrogen test which we have applied to influenza virus vaccine are summarized below: Weight Pre-injection temperature Post-injection temperature rise at 1 h at 2 h at 3 h Maximum rise Rabbit I 1 800g 39.60C 0.60C 0.60C 0.30C 0.60C Rabbit 2 2 000 g 39.20C 0.80C 0.80C 0.50C 0.80C Rabbit 3 2000 g 39.50C 0.50C 0.30C 0.20C 0.50C Pyrogen tests were also done using several different doses of the vaccines prepared for clinical studies sponsored by the Division of Biologics Standards. These were prepared from the monovalent A2/ Aichi/2/68 strain at several CCA concentrations, and the B/Massachusetts/3/66 strain was used as a control. All these vaccines were purified by zonal centrifugation and it appears that a dose of 0.1 ml per kg of body-weight is the most sensitive for this group of vaccines. As the CCA unitage of the A2/Aichi strain increased so did the reactivity as measured by this test. It should be pointed out that these vaccines were prepared from a single concentrate by means of 2-fold serial dilutions. In view of the findings reported by others it was surprising that the B/ Massachusetts strain vaccine was less reactive in this test than any of the concentrations of A2/Aichi. It is worth mentioning, however, that the lot used by Mostow et al. (see page 525) was produced by a earlier process. For comparative purposes, 4 commercial vaccines were chosen at random and subjected to the same type of pyrogen test, using 0.1 ml/kg of body-weight. Vaccine 1 was zonal-centrifuged and in order to have this vaccine at the same concentration, in terms of the human dose, as the other 3 vaccines it was diluted 1: 2 prior to the pyrogen test. Vaccines 2, 3, and 4 were concentrated by Sharples centrifuga- tion. All were monovalent, Hong Kong vaccines which were released during the last season. There is a striking difference between the reactiv- ity of the zonal-centrifuged and the Sharples- centrifuged vaccines as shown by the following 573 GENERAL DISCUSSION-SESSION III tabulation of the combined results tests: A2/Aichi vaccine * 200 (104) CCA/ml 400 (262) CCA/ml 800 (636) CCA/ml 1 600 (1 026) CCA/m1 2 400 (1 616) CCA/m1 B/Mass. vaccine * 800 (614) CCA/ml Commercial vaccines )f the 2 series of of Hong Kong influenza occurred. Vaccine had been given in most of these to children whose Mean maximum parents gave consent. The incidence of influenza temperature rise was the same in the unvaccinated and in those given 0.50C ** polyvalent vaccine. In only one of the few schools 0.50C ** given Hong Kong vaccine was there a statistically 0.60C significant reduction in incidence, and it may be 0.80C ** significant that this was the only school in which 0.8°C ** the epidemic started between a few days and a month after vaccination. In all other groups, in which no 0.40C ** effect was seen, the interval was a few days or 2-3 months. 1 0.20C 2 1.20C 3 0.60C 4 1.00C * The expected CCA content is shown first, followed by the assayed content in parentheses. ** Geometric means of results obtained in tests with doses of 1.6 ml/kg, 0.4 ml/kg, 0.1 ml/kg and 0.025 ml/kg. More experience with this type of testing, using a larger number of lots from each manufacturer, is necessary in order to assess its usefulness in deter- mining lot variability in terms of reactivity. The degree of correlation between the results of this test and reactivity observed in man remains to be determined. DR D. A. J. TYRRELL. It may be of value to summa- rize some points from a study of Hong Kong vaccine made in Great Britain by the Medical Research Council's Committee on Vaccination against In- fluenza and Other Respiratory Viruses. The ques- tion we wished to answer was whether vaccine given intramuscularly or intranasally would protect against natural infection. The vaccines used were made to the British standards and the Hong Kong vaccine contained 7000 haemagglutinating units. Studied were made in boarding schools in which laboratory-proved outbreaks occurred. In one the incidence was low, whereas in another it was higher, and there appeared to be a reduction in incidence in those given vaccine by both routes, but the trials were not double-blind. In a double-blind trial in which saline or polyvalent vaccine were used as placebos there was a school in which a high incidence (about 60%) of influenza occurred and there was no evidence of protection. Two studies were made in factories using a double-blind pro- cedure, but the incidence of disease was low and no firm conclusion could be drawn. We were provided with data concerning over 20 boarding-schools in which proved epidemics DR M. C. WILLIAMS. In a number of the papers presented there has been some evidence that vac- cines prepared with A2 strains appearing before the Hong Kong variant were protective during the recent epidemic. This has practical importance when production of vaccine to the new strain is inadequate. There have also been reports of the protective value of the zonally centrifuged A2/Hong Kong/68 vac- cine varying from 0 to 80%. Double-blind, random- ized, vaccine trials made between November 1968 and March 1969 among military personnel in Canada have bearing on these two points. The first was made in over 4000 military person- nel in the Maritime Provinces, using 2 zonally centrifuged Eli Lilly vaccines-B/Massachusetts/66 and A2/Hong Kong/68. From a comparison of the numbers off duty with respiratory disease in the two groups, it is estimated that the A2 vaccine gave at least 60% protection. The second trial was in Ontario, in nearly 6000 volunteers, using 3 formol-inactivated vaccines in routine production at the Institute of Microbiology, Montreal-namely, (1) A2/Hong Kong/68; (2) B/ Massachusetts/66; (3) A2/Montreal/68 and B/ Massachusetts/66. Using the same criteria of illness, it was found that both vaccines containing the A2 strains gave a similar protection, estimated at 60%, when compared with the vaccine B/Massachusetts/ 66. The A2/Montreal/68 strain was isolated in January 1968 and was in routine production as a vaccine before the Hong Kong strain was isolated. A careful comparison of the two A2 strains is needed. It would also appear that, in the absence of a vaccine specific to a new pandemic strain, it should not be assumed that vaccine made from recent strains will not give useful protection. DR J. H. S. GEAR. Although only limited amounts of inactivated vaccines could be produced quickly 574 GENERAL DISCUSSION-SESSION III in South Africa after the A2 strain became available in 1957 and the Hong Kong strain in 1968, both vaccines were shown to have some efficacy. Vaccine precipitated with aluminium phosphate gave some- what better antibody response than did vaccines without adjuvants. Since the Hong Kong strain epidemic did not begin in South Africa until March 1969, several million doses of vaccine from Europe and the USA were available. DR P. A. SMALL, JR. I should like to stress the local nature of the secretory antibody response. Our work with the respiratory system has shown that stimula- tion of the nasal cavity with influenza vaccine leads to nasal antibody but no sputum or salivary anti- body and little, if any, serum antibody. Stimulation of the lower respiratory tract leads to sputum IgA and serum IgG antibody but not to salivary or nasal antibody. Hence the response is quite local and if one is to attempt to produce protection by local immunization, all surfaces which are potential portals of entry should be stimulated. The second basic observation is that the respiratory secretory IgA immunological response is less specific than the humoral (IgG) response. Volunteers immunized with classic A2 virus demonstrated an approximately 8-fold rise in serum, nasal, and sputum virus-neutralizing antibody for this homo- logous virus. Assays using A0, A2/Hong Kong and parainfluenza viruses showed the pre- and post- immunization sera to have comparable titres and therefore no significant increase in antibody. Pre- and post-immunization analysis of the virus- neutralizing nasal and sputum antibody using para- influenza virus showed no difference, but there was a 3-4-fold rise when the A2/Hong Kong and AO viruses were used. Hence the antibody in the secretions was almost half as effective against these heterologous viruses as against the homologous ciassical A2. Finally, I should like to speculate as to a possible mode of action of influenza vaccines. Assuming that protection results from the presence of antibody in respiratory secretions, then the critical event would be the stimulation of the plasma cells under- lying the respiratory epithelium. This could be achieved by either local or parenteral administration (if some of the injected material found its way to the respiratory tract). However, the route of administra- tion would have a tremendous effect here. Specific- ally, intradermal administration of vaccine might lead to good serum antibody but not to as much secretory antibody. It will therefore be important to check protection as well as serum antibody for people immunized by the intradermal route. Other phenomena, such as higher pre-existing serum levels of antibody, might decrease the fraction of antigen reaching the respiratory tract and decrease the effectiveness of parenteral vaccination. This could conceivably explain the difference between our two studies which showed that in 1967-68 spray immuniz- ation with classical A2 vaccine (which had obviously been in circulation for some time) was much more effective than parenteral, whereas in 1968-69 parenteral was superior to spray immunization. DR R. B. COUCH. We administered various doses of inactivated zonal-centrifuged A2/Aichi vaccine to young adult prisoner volunteers. Each volunteer received a single subcutaneous injection and serum was drawn 4 weeks later for antibody determina- tions. With doses varying from 100 CCA units to 1800 CCA units there was an increasing frequency of responders and of magnitude of antibody response as assessed by haemagglutination-inhibition and neutralization. The increase was statistically sig- nificant. Reactions were low in all groups. Fifteen volunteers were given 1800 CCA units in a single nasal instillation and 13 were given the same quantity in a single subcutaneous injection. Four weeks later these individuals and 19 antibody-free controls were challenged with 3000 TCID50 of a Hong Kong strain by nasal instillation. A reduction in infection was noted in both vaccine groups but it was not statistically significant. Virus shedding patterns were also not significantly different from those in the controls. However, if the 3 individuals given vaccine intranasally and the 1 given vaccine subcutaneously who failed to develop serum anti- body were eliminated, then both vaccine groups exhi- bited less infection than the controls. The two vaccine groups were not different from each other. Nasal secretion antibody tended to parallel serum antibody. DR B. R. FORSYTH. Results similar to Dr Couch's have been obtained in studies with which I have been associated; the variation in responses reported at the session could well be related to the methods of local inoculation. The methods of administra- tion need study. DR W. P. EDMONDSON. We have recently evaluated inactivated influenza vaccine prepared by the Eli Lilly Company after subcutaneous administration 575 GENERAL DISCUSSION-SESSION III by jet gun or nasopharyngeal administration by DeVilbis Nebulizer 286. Medical students (100) and elderly hospitalized adults (2100) divided into 4 groups were given 400 CCA units of either A2/ Aichi/2/68 or B/Massachusetts/66 vaccine by gun, spray, or both methods. The spray was divided to give approximately 100 CCA to each nostril and 200 CCA to the oropharynx during inspiration. Sera were obtained from all students and 200 elderly persons before and 3 weeks after immuniza- tion and tested for HI and neutralizing antibody. With the spray vaccine, 19% of the elderly and 17% of the students with low or absent preimmuniz- ation HI antibody had 4-fold rises, and the group titre rose by 1 dilution. After subcutaneous injec- tion, 82% and 86% respectively had 4-fold rises, with an average final titre rise of 4 dilutions. Neu- tralizing antibody responses closely paralleled the HI responses. Double (gun and spray) vaccination appeared to confer no significantly greater benefit than vaccination by gun alone. An insurance company, whose 400 employees have had their illnesses recorded on IBM cards daily for more than 6 years and who were accustomed to nose and throat swab and serum specimens being taken at the time of illness, also volunteered for a vaccination programme without placebo. One-half received vaccine by the same 3 study routes, and all employees, vaccinated and unvac- cinated, were followed up by the routine surveillance customary in the company. Virus-positive Hong Kong illness occurred from 23 November 1968 to 17 January 1969. Of the total, between 450% and490% reported symptoms of illness, 62%, 66% and490% of such reports being from the jet-injection vaccine, spray vaccine, and unvaccinated groups respectively. A2/Hong Kong-positive serum speci- mens were obtained from 5%, 16% and 20% of these respective groups, thus showing a protective effect from jet-injection vaccine ranging from 69% to 75 %. Similarly, 1.1 %, 2.1 % and 2.1 % of work days were lost by the respective groups, suggesting that jet-injection vaccine reduced lost work days by as much as 50 %. When febrile illnesses only were tested in the laboratory (83 %, 69% and 62% of the respective groups), attack rates for A2/Hong Kong of 3%, 13% and 11% were obtained for the jet- injection, spray and unvaccinated groups, indicating a protection of from 73% to 75% by parenteral immunization. In the medical students there was a 71 % reduction of symptoms, a 600% reduction in all illness, and no febrile illness proved to be related to A2/Hong Kong virus after parenteral vaccine. In the elderly patients the protective effect over-all for the paren- teral vaccine was between 55% and 60%. It was concluded that I nasopharyngeal dose of killed A2/Hong Kong vaccine neither produced significant antibody levels nor protected individuals from natural influenza infection but that the vaccine was effective when administered subcutaneously. DR J. L. SCHULMAN. Dr Reed Dunnick in our laboratory has compared the effects of intranasal and parenteral administration of inactivated Aichi vaccine in mice challenged with Aichi virus. One intranasal dose of vaccine did not result in reduced pulmonary virus titres following challenge, but did potentiate the protective effects in mice previously immunized parenterally or intranasally. Two parenteral doses of vaccine provided the greatest protection and in addition the group so vaccinated was the only one in which antibody was detectable in bronchial secretions. Therefore, intranasal ad- ministration of inactivated vaccine may be a less efficient method of eliciting a local antibody response than parenteral administration of the same vaccine. Furthermore, in answer to the question raised by Dr Small about a possible difference in the immun- izing effectiveness of intranasal vaccine in immuno- logically prepared and unprepared subjects, our studies in mice demonstrated that intranasal Aichi vaccine had little effect in animals previously immunized parenterally with A2/Japan/305/57 virus, whereas parenteral administration of Aichi vaccine to animals previously immunized parenterally with the A2/Japan/305/57 virus was associated with a definite booster effect, resulting in more protection than that observed in animals immunized only with the Aichi vaccine parenterally. In other words, with an antigenically new virus intranasal immuniza- tion may be particularly inefficient. DR P. VON MAGNUS. In studies carried out 10 years ago with A2 strain vaccine, fairly good antibody responses were obtained after intradermal inocula- tion of the lower doses used. There was no indication of a higher antibody response to the first intra- dermal inoculation but the results after the second inoculation indicate that 10-40 CCA units given intradermally may elicit an antibody response similar to that produced by 100-200 CCA units given subcutaneously. 576 GENERAL DISCUSSION-SESSION III 577 DR S. R. MOSTOW. Dr Tauraso presented data which compared the intradermal and subcutaneous routes of administration of influenza vaccine. The best homologous HI antibody response, however, was a 7-fold increase in geometric mean titre achieved in one group. In work done at the National Communicable Disease Center among prison volun- teers, up to 4800 CCA units were administered sub- cutaneously and up to a 35-fold increase in homo- logous geometric mean titre was achieved. In addition, the higher doses were associated with more broadly reacting antibody and the appearance of type-specific neutralizing antibody in nasal secre- tions. Vaccine efficacy (i.e., disease prevention) should be our aim and, as suggested in earlier presentations, protection may be dependent on the presence of specific antibody in respiratory secre- tions. Our data suggest that the antigenic mass given determines whether or not respiratory anti- body appears. Therefore we should not easily be swayed into accepting the intradermal route of administration of influenza vaccine without appro- priate efficacy studies. DR W. R. DOWDLE. During this session several investigators have presented data from studies in which serological results were employed as a basis for determining vaccine efficacy. Twelve years ago, Rapmund and associates b demonstrated that upon natural challenge of immunized individuals with high antibody titres many of the men became ill and virus could be recovered readily. Many of these men did not demonstrate a further 4-fold antibody rise in either CF or HI tests. Although some investigators may question these results, the findings of Rapmund and his associates can hardly be igno- red. Until these findings can be disproved, antibody response alone cannot be used as a measure ofefficacy (see the paper by Schoenbaum et al., page 531). b Rapmund, G., Johnson, R. T., Bankhead, A. S., Herman, Y. F. & Dandridge, 0. W. (1959) U. S. armed Forces med. J., 10, 637-649. 17