Experience with the production of Hong Kong influenza vaccine at the Statens Seruminstitut, Copenhagen

556 DISCUSSION PAPERS-SESSION III Experience with the Production of Hong Kong Influenza Vaccine at the Statens Seruminstitut, Copenhagen by PREBEN VON MAGNUS a During the winter of 1968-69 the influenza department of the Statens Seruminstitut in Copen- hagen produced about 200 000 doses of Hong Kong influenza virus vaccine. The following is a brief discussion of some of our experience with the cultivation of this strain in eggs and with the concentration and purification of virus. The strain we used was obtained from Stockholm, where it had TABLE I HA TITRES IN ALLANTOIC FLUIDS AND SHARPLES 60-FOLD CONCENTRATES ATTAINED IN THE STATENS SERUMINSTITUT WITH 10 REPRESENTATIVE SAMPLES OF A2/Hong Kong/68 VIRUS Batch HA titre (logio) no. | Allantoic fluids Concentrates 314 3.62 5.22 316 3.05 4.90 318 3.58 4.92 320 3.16 4.81 322 3.58 5.00 324 3.16 4.74 326 3.32 4.87 328 3.05 4.72 330 3.62 4.79 332 3.88 5.10 Geometric mean titre 3.40 4.90 been partially adapted to eggs by 12 allantoic passages in the National Bacteriological Laboratory. The first egg passages in our laboratory were low in titre but after 4 passages the titre suddenly increased by 1 log and remained high in the sub- sequent 2 passages. Allantoic fluid from the 6th passage had a titre of 103.7; this is comparable to that obtained with well adapted influenza strains by the haemagglutinin (HA) method employed in our a Director, Statens Seruminstitut, Copenhagen, Denmark. laboratory. The 6th passage fluid was stored at - 60°C and used as seed for batches ofabout 500 eggs each. The allantoic harvests for vaccine production were pooled in batches of 10 litres and, after clarifica- tion, were concentrated in a Sharples centrifuge. Table 1 shows the haemagglutinin titres of 10 representative Hong Kong batches. The middle column shows the titres of the allantoic fluids, the right column those ofthe corresponding concentrates. The titres of the fluids will be seen to vary con- siderably from batch to batch; they are also fairly low, the geometric mean titre being 103@. This is only a little more than half the titre obtained with well adapted influenza strains of previous years. TABLE 2 HA TITRES IN ALLANTOIC FLUIDS AND SHARPLES CENTRIFUGATION FRACTIONS ATTAINED IN THE STATENS SERUMINSTITUT WITH REPRESENTATIVE SAMPLES OF ASIAN, B AND HONG KONG INFLUENZA VIRUSES HA titre (logio) A2/ B/ A2/Hong Kong/68 Singa- Joh- pore/57 annes- Prepn Prepn Prepnpore aJuburg 1 2 3 Allantoic fluids(clarified) 3.63 3.56 3.58 3.32 3.47 Effluent liquids: Allantoic fluid 2.36 2.66 2.08 1.68 2.01 Saline, 5 min 2.36 1.66 1.86 1.53 1.83 Saline, 10 min 1.13 1.20 0 0 0 Saline, 15 min 0 0 0 0 0 Sharples concentrate: a Supernate, 160 ml 5.31 5.21 5.11 4.80 4.80 Pellet in 5 ml 4.96 5.21 5.33 5.40 5.48 Total HA units in: Allantoic fluids 7.63 7.56 7.58 7.32 7.47 Final concentrate 7.51 7.41 7.31 7.00 7.00 HA recovery rate [76% 71 % 54% 48% - 38% a Sharples sediment resuspended in 160 ml saline and cen- trifuged at 1500 rev/min for 30 min. 2404D PRODUCTION OF HONG KONG INFLUENZA VACCINE AT STATENS SERUMINSTITUT 557 As could be expected, the low titres of the allantoic fluids are reflected in the concentrates; the mean titre of these is I0"-" which is less than half of what is usually obtained with other influenza strains. The low virus content in the concentrate is mainly due to the low titre in the allantoic fluids, but low recovery of virus in the final concentrate is a con- tributing factor. Only about 50% of the original HA activity was recovered in the concentrate. This is in contrast to results obtained with previous influenza strains, where the recovery rate has consistently been at least 70%. An analysis of the losses during centrifugation is shown in Table 2, which records the haemagglutinating activity in the allantoic fluids and in the Sharples centrifugal fractions for various vaccine preparations. Two columns show representative results as obtained with Asian 1957 virus and the B/Johannesburg strain. To the right are shown results obtained with 3 different batches of Hong Kong virus. The recovery rates of haemagglutinins in the final concentrate are listed in the bottom row. The recovery rates with the 3 Hong Kong preparations were only 54%, 48% and 38%, respectively, as compared with a recovery rate of more than 70% with both the Asian and the B strains. Virus antigen is lost during centrifugation with the effluent liquids, but the loss is insignificant with all strains, and, if anything, it is lower with the Hong Kong virus than with the other strains. Some anti- gen is also lost in the pellet after the final low-speed centrifugation. This loss is very small with the Asian and B strains, but surprisingly high with all 3 preparations of Hong Kong virus. It appears likely that the comparatively poor recovery of Hong Kong virus in the final concentrate may be due to an aggregation of viral antigen which is sedimented and removed at low gravity. In summary, the production of Hong Kong influenza vaccines was carried out at the Statens Serumrrinstitut by conventional methods without any particular difficulties, but with a relatively poor yield. This was due mainly to low titres of the allantoic fluids and partly to a lower than usual recovery of virus by differential concentration. Most likely the adaptation to eggs of the Hong Kong virus strains we used was insufficient. I know of other smaller vaccine production units which have had similar experiences. For such smaller laboratories it would undoubtedly be a great advantage if the adaptation of new strains could somehow be centralized (for instance by the World Health Organization) so that virus could be made available which had been sufficiently adapted to eggs to be suitable for the preparation of seed virus for vaccine production. Immunogenicity of Purified and Conventional Inactivated Influenza Virus Vaccines by WILLIAM J. MOGABGAB a Observations were made in the fall of 1968 on 2 purified influenza virus vaccines, one derived from calf kidney cell cultures and ether-treated after density-gradient centrifugation, and the second prepared with chicken-embryo-propagated viruses and purified by zonal centrifugation. Both contained 600 CCA units per O.5-ml dose, of which 300 CCA units were of 1962 and 1964 influenza A2 strains and 300 CCA units were of 1966 influenza B. When these preparations were administered to adults sub- a Section of Infectious Diseases, School of Medicine, Tulane University, New Orleans, La. 70112, USA. cutaneously, systemic manifestations were very uncommon and local reactions to both were quite mild. As shown in Table 1, haemagglutination- inhibition (HI) antibody responses to the viruses contained in the vaccine as well as those to the 1968 influenza A2 strain were similar after either vaccine. Larger incremental changes occurred in those individuals with lower prevaccination antibody titres. An unexpected finding was the lower titres, and of more significance in evaluating antigenicity of influenza vaccines were the smaller increments, obtained with the micro technique. 2404E