DTIC ADA259295: Laboratory Tests And Reference Reagents Employed in Studies of Inactivated Hepatitis a Virus Vaccine

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4. TITLE AND SUBI3TTLE.........
T.ABORA¶IORY TE.STS AND REFERENCE RENGEN91, EMPLOYED III
STUDIES OF INACTIVATED HEPA~TITIS A VIRUS VACCINE WRGC
6. AUTHOR(S)
L.N. Binn, P.O. IlacArthy, R.H. marchwicki, M.H. Slogjren,
C.H-. Hoke Jr., J.R. Burge and E. D'I-ondt
7W. APLERTFEORRM IRNGE EODR GAARNMIZYA TIOINN SNTAIMTI(UST) EA NOD FA ORUSE(S)PERAEPROCr T RPOT NUUMSEORGNZTO
BLDG. 40 RM 2046
14th & DIHLIA ST. N1
DEPARTMENT OF VIRUS DISCYNSE~S
9.SOSOIGjMOIOIG GNYNAME (S) AND ADO SS 5) ¶0. SPONSORING/IMONITOP.ING
~. PONORNGMAOGITNOCI N AGENCY REPORT NUMBER
11. SUPPLEA ;d.,ARY NOTES
128. OISTR1IIaCN/4A'AILABILITY STATEMENT 12b. DISTRIBUTION COOE
APPROVED FOR PUBLIC RELEASE: DISTRIBUTION IS UNLIMITED
13. ASSTRAC' rm 2O00wofds)
Procedures to evaluare inactivated hepatitis A vaccines in volunteers have been e'xamlined.
So/id-phase ininuinuassav merte standardi:ed wiitlh reference preparations and havc hee?
tested to fll('Ourc unith 1 di resrnons' to imnmuni--ation and antwgen conltenlt (11 t'Uc~line'
Folloio ng iniownuatiw,ti .inerei taus a good correlation betiveen an tihodvi rc4SponN(c detter-
mnined ivith cumnnerewia inununoussa vs. and neutrali~atton tires. ask measured At 'im
radnninmunotfocus inhibition lest. Hostever, at lowter tlres of neutrali:ing antiihod . thfe
comm~ercial Jimunlultassai (4 ten vitched negativ esut.T mrv h sensitivit i of ihu
inininwassa'l the serumn volume hvas increased. A /ou Iod inrain t~ eu resulted
in greater senszrivit 1. increasing/Iroin 54 to 94%. ithiI'e retaining 100",;~T ecificiti. further
i. ncreases in the volume of test seruml resulted in a loss of specificity. In a comparison 01
neuirali:atw,: tests, si milar itares of post vaccination serums wiere obtained bi using the
HMI 75/ 1Sf citopathic strain of hepatitis A vir..s in a plaque reduction assai or the
HM 175 parental virus in the radioinimunofocus inhibition test. U~se of the crvwputhic virus
obviates the need for radioociivel'v labelled serum and reduces the time takeni to conduct
neu~tralization tests. The current laboratorY procedures can meet the needs of large field
trials of inacuvaied hepatitis A vaccines.
14. SUBJECT TI IS, NUMBER OF PAGES
Hepatitis A vaccine, VAC V25 4
16. PRICE CODIE
I
17. SECURITY F ATIOE 18, SECURITY CLASS IfICATION I9. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRAC
OF REPORT OF THIS PAGE OF ABSTRACT
IUNCLASS1
U1CLA.SS UNCTUIZ5 T
NSN 'S4O0-'*2SC
Laboratory tests and reference reagents
employed in studies of inactivated
hepatitis A vaccine
L. N. Binn&*. P. 0. MacArthv*. R. H. Marchwicki*. M. H. Sjogren*.
C. H. Hoke Jr.*. J. R. Burge* and E. D'Hondtt -
Procedures to evaluate inactivated hepatitis .4 vaccines in volunteers have been examnined.
Solid-phase iniunnoassavs were standardized with reference preparations and have been
tested to measure antibody response to immunization and antigen content of vaccines.
Following immunization, there was a good correlation between antibhdv response, deter- 6I_ý__
mined with commercial immnunoassays. and neutralization titres. as measured bh the
radioimnunofo'cus inhibition test. However. at lower titres of neutralizing antibodyv, the
commercial imnmunoassav often yielded negative results. To improve the sensitivity of the JI M
imnmtunoassay, the serum vohine vas increased.A.4f uriolh increase of test serutm resulted
in greaters ensitivity, increasing.from5 4 to 94%, while retaining 100% specificity. Further
increases in the vohlme of test serunm resulted in a loss o/ spec'ilcit., In a comparison of"
neutralization tests, similar titres of postvaccination sera wtere obtained by using the
1TM175 181 ctopathic strain of hepatitis .4 virus in a plaque reduction assay or the
HM 175 parental virus in the radioimmunolocus inhibition test. U.w of the cytopathic virustýW
obviates the need for radioactively labelled se'rum antir ehuces the timne taken to conduct o
nmeutrclization tests. The current laboratory procedhures can meet the needs of largeefield
trials of inactivated hepatitis A vaccines.
Keywords: Hepatitis A: inimunoassav: neutralization test
INTRODUCTION rely small numbers of serum specimens. Therefore.
attempts were made to increase the sensitivity of the
Solid phase immunoassays (SPIA) have made vital con- immunoassavs" and results are summarized in this
tributions to the control of hepatitis A. Initially. the report. To enable greater use of the neutralization test a
antibody assays were used with a high degree of sensiti- cytopathic strain of hepatitis A virus (HAV) was also
%itv and specificity for diagnostic and epidemiological evaluated for use in determining the neutralizing anti-
purposes. This was due to the rapid. high and sustained body response to inactivated vaccines. In addition, this
antibody response following infection'. During studies report briefly summarizes use of SPIA for measuring the
on viral cultivation, antigen assays were employed to antigenic content of inactivated vaccines.
detect propagation and estimate virus concentration-2.3.
Immunoassavs for antigen and antibody were extensi-
vely used during the development of inactivated vaccines MATERIALS AND METHODS
and enabled rapid determination of viral antigen content
and antibody response4. In contrast to natural infections. Antigen assay
the antibody responses to the initial inactivated vaccines HAV antigen content was determined by a solid phase
were modest and. at times, results of commercial immu- radioimmunoassay (SPRIA) and by enzyme-linked
noassays were negative although neutralizing antibody immunoassay (EIA) as previously described7". A cali-
was presentc. In addition, the radioimmunofocus inhibi- brated reference preparation was used to determine the
tion test (RIFIT) is a highly labour-intensive neutraliza- antigen contentx.
tion test requiring 10-14 days to complete for comparati-
Serum specimens
"Divisions of Communicable Disease and Immunology and
Biometrics, Walter.Reed Army Institute of Research. Wash-
ington, DC 20307, USA. SmithKline Beecham Biologicals, used from volunteers in previous institutionally
Rixensart, Belgium. §To whom correspondence should be approved protocols5.The volunteers had received two or
addressed more doses of inactivated HAV vaccine produced at
S0 Vacie 0264-41OXi92J10061)2-4
,7. / C, 1992 Butterworth-'llfltflaflf Ltd
Testing inactivated hepatitis A vaccines: L. N. Binn et al.
Walter Reed or SmithKline Beecham Biologicals (Rixen- 120
sart).
10 0
Immunoassay 8 " C
The commercial RIA (HAVAB, Abbott Laboratories, •
Chicago, IL, USA) was used. Ten pl1te st serum and 200 600
ip1 competing labelled serum were used in the standard .
assay and modified tests were done with combinations of 40 *-
20 and 190 l.l, 40 and 170 pl, and 80 and 130 pl, of test SE
serum and competing labelled serum respectively. A 20 0
World Health Organization (WHO) reference hepatitis .
A immune serum globulin was used for control 0
purposes9.
--20 I i I I I
- 1:10 1:160 1:2560 1:10 1:160 1:2560 1:10 1:160 1:2560
Neutralization test 1:40 1:640 1:40 1:640 1:40 1:640
The RIFIT was used to titrate neutralizing antibody'°. Neutralization titre
For a neutralization test with the cytopathic HlM 175/18f Figure 2 Relationship of the neutralization titres and standard and
strain, virus was propagated in FRhK4 cells and the foci modified RIA for HAV antibody in vaccine recipients. Test volume: a. 10
were detected by staining with crystal violet' ;lb, , 20 Al; c, 40 Al
Statistical analysis Table 1 Modified hepatitis A virus antibody RIA: percentage inhibition
The regression approach to analysis of variance was before and after inactivated vaccine in responding volunteers
used to test for parallelism of test groups'3. Serum tested Mean inhibition (%) and No. seropositive/total
with test serum at
RESULTS AND DISCUSSION 10 Al 20 pl 40 Al 80 Al
Vaccine antigen Pre-immunization -12.5 -8.6 -5.7 23.1
(0/8) (0/8) (0/8) (0/8)
Both SPRIA and EIA have been used to detect and Postimmunization 54.3 64.4 72.6 85.7
measure HAV antigen content. The amount of antigen (5/8) (718) (8/8) (8/8)
can be readily estimated by comparison with a standard
curve (Figure 1)6-8. Slopes of dilution curves for crude Neutralization titres ranged from 1:10 to 1:160, GMT = 1:40. With increas-
harvest, purified and formalin-treated preparations were inmv olume of test serum the competing labelled serum was decreased
similar to that of the reference preparation. Formalin to keep volume constant (210 A.l)
inactivation did not change reactivity. Results of statisti- to determine the HAV antigen content on alum-
cal analysis for parallelism of the slopes of the four Xiasorbed vaccines.
preparations supported this conclusion [f value of 2.54
with 3 and 9 degrees of freedom (p=O.12)]. Vaccine immunogenicity
At present, each laboratory is employing its own refer-
ence preparation for which it has determined the antigen Initial observations of antibody response to inacti-
content. However, there is a need for a reference vated HAV vaccines indicated that there was a correla-
standard HAV antigen preparation for comparative and tion (r = 0.83) between the neutralization titre and the
regulatory purposes. Studies for this purpose are being percentage inhibition using standard commercial RIA
., sponsored by the WHO. In addition, methods are needed which employs 10 pi of serum (Figure 2). However, many
serum specimens with neutralization titres of < 1:160
316 had RIA results below the minimum positive value of
50% inhibition. To improve the sensitivity of RIA.
increased volumes of serum were tested from volunteers
100 before and after immunization (Table 1). For this
purpose, serum specimens with low titres of neutralizing
31.6 antibody (1:10 to 1: 160) were selected and the percentage
.oinhibition was calculated by using 10 pi of the negative
-
control serum supplied by the manufacturer. There was a
10 - progressive increase in percentage inhibition with greater
volumes of test serum, which was in part due to the
3.16 decreased amount of labelled competing serum. How-
ever, none of the eight pre-immunization serum speci-
mens yielded positive results when the test volume was
1:320 1:100 1:32 1:10 increased. After immunization, the number of seropos-
1.1000 0itive specimens increased from five (with the standard
Dilution of HAV antigen test volume of 10 pI) to all eight (with 40 or 80 p1 test
Muire 1 Radloimmunoasaay for quantitative HAV antigen determi- serum).
nation. 0, Standard; 7, harvested HAV; n, purified HAV: 0>, formalin- To test specificity, standard and modified RIA was
"InactivatedH AV. PIN, Poeltive/negative carried out with serum specimens from six vaccinated
Vaccine, Vol. 10, Suppl. 1, 1992 S103
Testing inactivated hepatitisA vaccines: L. N. Binn et al.
Table 2 Modified hepatitis A antibody RIA: percentage inhibition before with few exceptions. At both 10 and 20 p. test volumes.
and after administration of inactivated vaccine in volunteers who did not one additional positive was detected and, at 40 p1, there
develop neutralizing antibody was one less. Analysis (Figure 2) of the data indicated
Serum tested Mean inhibition (%) and No. seropositive/total that the slope of the relationship between neutralization
with test serum at titre and percentage inhibition was unchanged except
that the Y intercept had shifted to the left, indicating
10lapl 20111 401 1l 80 1l greater sensitivity. Statistical analysis of the data, using
Pre-immunization 9.4 19.9 23.4 32.1 analysis of variance"3, supported the conclusion of a
(0/6) (0/6) (0/6) (0/6) common slope (f = 1.29 and p = > 0.05).
Postimmunization 18.6 23.6 38.3 57.0 The sensitivity of standard and modified RIAs to
(0/6) (0/6) (0/6) (5/6), identify sera with HAV neutralizing antibody from
Neutralization titre < 1:10 volunteers given inactivated vaccine increased from 52%
"-2/6s eropositive when calculated from same volume of pre-immuniza- with 10 pl test serum to 94% using 40 pl, with 100%
lion serum specificity (Table 4). However, the specificity fell to 17%
wi(cid:127)h 80 V1t est serum. Use of the homologous pre-serum
value for 80 pl serum increased the specificity to 67%. a
Table 3 Modified hepatitis A virus antibody RIA: evauation in 43 volun- level that was still unacceptable. The findings indicate
teers given inactivated vaccine that modified RIA employing fourfold increased
Serum Neutral- No. seropositive/total and Inhibition volumes of serum (i.e. 40 pl), achieved significantly
ization (%), with test serum at increased sensitivity without loss of specificity and can be
titre performed in a conventional test.
10toP 20 1 40lil Recently, a cytopathic variant of HAV, HM175118f,
has beer described which retained normal antigenicity
PProes-tiimmmmuunniizzaattiioonn 00 00//453(2(1.4.1)) 00//453(2 (.07.)8 ) 00//453(1(102.5.3) ) and had a more rapid rate of replication' 2.. NNeerturathria1ilni zingg
10 0/6(15.5) 0/6 (34.2) 4/6 (56.9) antibody titres were similar with HM 175/18f in plaque
40 1/8 (34.9) 3/8 (48.3) 7/8 (63.2) reduction neutralization tests (PRNT) and with parental
160 519(51.9) 7/9 (66.3) 9/9 (81.7) HM 175 virus using RIFIT (Figure 3). 0f146 postvaccina-
640 10/12 (54.4) 12/12 (75.4) 12/12 (86.4) tion serum samples tested using RIFIT and PRNT titres
2560 3/3(81.4) 33 (89.0) 3/3(92.6) of > 1:10, 18(39%), had the same titre in both tests and
Neutralization titre an additional 23 (50%) had titres within fourfold. Thus,
for RIA positive 160 62 <10 nearly 90% of the positive sera had titres that were
-Mean inhibition calculated with 10 pi negative reference serum
Table 4 Sensitivity and specificity of standard and modified AlAs for
hepatitis A virus neutralizing antibody in volunteers given inactivated
volunteers who did not develop detectable neutralizing vaccine
antibody (Table 2). As observed above, none of the six
pre-immunization serum specimens yielded positive Volume serum Sensitivity Specificity
results although the percentage inhibition increased by _- No. p to
nearly 25% with the eightfold increased volume of test
serum. However, five of the six postimmunization serum 10 26/50(52) 11/11 (100)
specimens had positive results, but only at the eightfold 20 3W/50 (72) 11/11 (100)
increased volume and when percentage inhibition was 4800 4172//1520 ((9140)0 ) 111//161 (1(71)0,0)
calculated by using the results with 10 p1 reference serum. 80 _12/12(100)____ _____
By using the value from 80 plI homologous pre-immuni- All pre-immunization sera were negative at each volume tested. Postim-
zation serum to calculate the percentage inhibition, the munization serum with neutralizing antibody titre of > 1:10 compared
(cid:127). number of positives was reduced from five to two. These with 10 pI reference negative serum in RIA
findings suggested that modified RIA with 80 pI test 4416 (67%) when calculated with 80 pi homologous pre-serum
serum would lose specificity but that volumes of < 40 pi
would achieve increased sensitivity without loss of speci- z 2560 - 3
ficity. - -
Further evaluation of modified RIA was carried out E 640 - 2 . 3
with serum specimens from 43 vaccinated volunteers . ,-
who had neutralization titres ranging from < 1:10 to
1:2560 (Table 3). None of the 43 pre-immunization 1.-
serum specimens had positive RIA results with any ,
increased volume tested. There was a clear relation Z 40 7 3
between the neutralization titre and the number of post- '2
vaccination serum specimens that were RIA-positive. 10 1
The mean 50% RIA conversion point was lowered from * : I
neutralization titres of 1:160 for the standard RIA using 1o 120 40 160 640 2560
10 pi to < 1:10 for modified RIA in which 40 pl serum
wSa1s6 t-efsoteldd,. thereby increasing the sensitivity of RIA by Radloimmunofocus inhibition test (RIFIT)
By using the same volume of homologous
pre-immunizatiof serum instead of thlvengtv control FIgure 3 Comparison of the radioimmunofocus inhibition test and a
calculate therpercensteage inhibititoh e n umtrol plaque reduction test for neutralizing antibody in vaccine recipients
serum to calculate the percentage inhibition, the number using pre- and postimmunization sera. 0, No. of individuals with the
of seropositives was unchanged at each volume tested, same itre in both tests
8104 Vaccine, Vol. 10, Suppl. 1, 1992
Testing inactivated hepatitisA vaccines: L. N. Binn et al.
within fourfold, reflecting a single test dilution: the evaluation of hepatitis A vaccines and will continue to
remaining five (I1%) had a 16-fold difference. The geo- play a vital role in vaccine production and use.
metric mean titre (GMT) for RIFIT was 1:100 and for
PRNT it was 1:72. All but one of the 53 pre-immuniza-
tion sera were negative in both tests. The exceptional ACKNOWLEDGEMENTS
serum was reactive at a dilution of 1:10 using RIFIT. The opinions or assertions contained herein are the
Thesc results clearly indicate that PRNT with HM175/ private views of the authors and are not to be construed
l8f could replace RIFIT and eliminate the use of radioi- as official or as reflecting the views of the Department of
sotopes and thus achieve considerable savings in time the Army or the Department of Defense.
and labour. The authors are grateful to Dr John Ticehurst for his
It will be important to evaluate the practice of using most helpful review of the manuscript and to Dr Douglas
the WHO reference immune globulin as a standard prep- Tang for statistical advice and assistance.
aration for quantifying antibody responses (expressed as (cid:127), US Government.
mIU ml) mnv olunteers given inactiv3ted vaccine. Errors
may result from comparing a late immune response to
infection with that to an inactivated purified vaccine at REFERENCES
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Vaccine, Vol. 10, Suppl. 1,1 99 S105