Immune response in healthy volunteers vaccinated with BCG plus killed leishmanial promastigotes: antibody responses to mycobacterial and leishmanial antigens

Immune response in healthy volunteers vaccinated with BCG plus killed leishmanial promastigotes: antibody responses to mycobacterial and leishmanial antigens Claire E. Sharpies*, Marie-Anne Shaw*, Marianella Castes*, Jacinto Convit t and Jenefer M. Blackwell *~ Antibody (IgG) responses to mycobacterial ( BCG; PPD, Mycobacterium leprae soluble antigen, M L S A ) and leishmanial (Leishmania mexicana L V4) antigens were measured in 208 initially PPD and leishmanin skin-test negative volunteers divided into four vaccine groups asJollows: 68 received BCG plus killed promastigotes (group A ), 47 received BCG alone (group B), 47 received killed promastigotes alone (group C), and 46 formed the diluent control (placebo, group D). Three vaccine doses were administered at 8 12 week intervals. Vaccinees were bled immediately, prior to each vaccination, and again at 3- and 12-month jollow-up. Skin tests were performed prevaccination, and again at the 3- and 12-month follow-up. Anti-BCG, anti-PPD and anti-MLSA IgG levels increased significantO' in groups A and B receiving BCG. The presence of leishmanial antigen (with BCG) in the inoculum suppressed the IgG response to Mycobacterium tuberculosis/ Mycobacterium bovis-related (PPD and BCG), but not M. leprae-related ( M L S A )related, antigens. A small but significant increase (relative to prevaccination level) in response to MLSA, but not to BCG or PPD was observed in the non-BCG-vaccinated groups. The background level of response to mycobacterial and leishmanial antigens was higher in the Venezuelan vaccinees than in non-endemic (British) volunteers. Responses to leishmanial antigen were not enhanced in the two vaccine groups receiving killed promastigotes (with~without BCG) compared with the BCG alone and placebo groups. Instead, all vaccine groups showed a pattern o# response consistent with either (i) a response to the skin-test antigen or, more likely, (ii) seasonal endemic exposure to leishmanial antigen. Interestingly, this endemic resl~onse to leismanial antigen was enhanced in the vaccine groups receiving BCG, despite the .fact that group B received no leishmanial antigen in the vaccine inoculum. When prevaccination IgG levels (mean + 3 standard deviations) were used to determine a negative cut-offl a low percentage ( < 38%) oJvaccinees converted to responder status for either anti-mycobacterial or anti-leishmanial responses, and those who did would be classified as 'low-re.sponder' status compared with titres observed in severe jorms of disease. Hence, although there was evidence .[or a background endemic response to both leishmanial and mycobacterial antigens, there was no evidence that vaccination per se led to a potentially disease exacerbatorv level of TH2-associated antibody response especially with respect to the anti-leishmania] response. Taken together with our earlier report that a high proportion of vaccinees, particularly in the BCG plus killed promastigotes group (> 90%), converted.~r T-cell responder phenotypes (skin test; proliferative response, interferon-7 production) to leishmanial antigens, these results indicate that this vaccine is" potentially protective .#or the majority of t~accinees. The results ~?/"this small trial thus provide a level of confidence in extending the protocol to test the £~'cacy oJ" BCG plus whole killed parasite vaccines in preventing disease. Keywords: Leishmaniasis; BCG; killed promastigotes *University of Cambridge Clinical School, Department of Medicine, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. tlnstituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, APDO 4043, Caracas 1010A, Venezuela. +~Towhom correspondence should be addressed. (Received 3 December 1993; revised 18 March 1994; accepted 7 April 1994) 0264-410X/94/15/1402-11 1994 Butterworth-Heinemann Ltd

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Vaccine 1994 Volume 12 Number 15

Antibody responses to mycobacterial and leishmanial antigens: C.E. Sharpies et al.

Vaccination against leishmaniasis using viable parasites or crude killed promastigote antigens has been carried out over many decades. In the Old World a procedure termed 'leishmanization' was employed (reviewed in Ref. 1) which involved inoculation of viable parasites to cause a lesion in a preferred body site. In the New World, crude whole parasite vaccines were tested against American cutaneous leishmaniasis (ACL) in Brazil in the late 1930s and early 1940s 2'3, and again more recently by Mayrinck and co-workers 4-8 using a cocktail of sonicated promastigotes from five or six strains of Leishmania. The results of these trials have shown a reduced incidence of disease among skin-test converted individuals, but not the complete protection that it is possible to achieve with either crude 9'1° or defined antigen ~,~2 vaccines in murine models of the disease. More recently we reported ~3 on a small trial carried out in Venezuela to determine whether the use of viable BCG as a co-stimulant with killed leishmanial promastigotes could enhance the immune response to leishmanial antigen, either through its action as a non-specific immunostimulant or due to cross-reactivity between mycobacterial and leishmanial antigens. This seemed a logical extension to the observations of Convit and his colleagues ~4'1s that BCG plus killed promastigotes was highly effective as an immunotherapeutic treatment in patients already presenting with cutaneous leishmaniasis. Before proceeding with large field trials to test the efficacy of the vaccine in reducing the incidence of disease, this smaller trial was established to ascertain whether a potentially protective immune response could be induced. Studies in mice 16"a7 and humans ~8 22 had all pointed to a crucial role for interferon-?-producing CD4 + T-helper 1 (TH1) cells in protection and cure of cutaneous leishmaniasis, usually associated with a strong delayedtype hypersensitivity skin-test response in vivo. Nonhealing responses are associated with IL-4/IL-5 producing C D 4 + T-helper 2 (TH2) responses 2°-22, normally characterized by high circulating IgG levels to leishmanial antigens 23-25. BCG is known to induce strong skin-test and interferon-7-producing T-cell responses 26, but it was not known whether co-injection of BCG with leishmanial antigen could also bias the Leishmania-specific immune response towards a T H I type response. In our earlier paper 13 we reported on cell-mediated immune responses measured in vaccinees. Although a potentially protective interferon-7-producing T-cell response was observed in a high proportion of vaccinees, it was important to establish whether the protocol had also elicited a potentially disease exacerbatory response in others. Here we report on antibody responses to mycobacterial and leishmanial antigens and discuss these in relation to T H 1 versus TH2 directed pathways in determining immune response to vaccination. MATERIALS AND METHODS

induration) to participate in the trial. This sample size was not intended to allow evaluation of efficacy of vaccination in terms of natural incidence of disease. The trial was established specifically to determine the effect of vaccination on the cellular 13 and humoral immune response. The rationale for choosing double-negative individuals was an attempt to identify individuals who were naive in terms of previous exposure to the vaccinating antigens. At the start of the trial, 68 vaccinees received BCG plus killed promastigotes (group A), 47 received BCG alone (group B), 47 received killed promastigotes alone (group C), and 47 formed the diluent control (placebo, group D). For clarity in presentation of the results, a diagrammatic summary of the vaccine trial protocol is presented in Figure 1. Throughout this paper, the vaccine groups will be refered to as A, B, C and D, and the timepoints for sampling as times 0, 1, 2, 3 and 4. The numbers of vaccinees remaining in the trial at times 3 and 4 respectively were: 63 and 54 for group A, 39 and 33 for group B, 38 and 29 for group C, and 44 and 35 for group D. Sex ratios (female:male) in the initial sample were 46:22 (group A), 36:11 (groups B and C), and 34:12 (group D). Ages were in the ranges 15 52 years (group A), 15-55 years (group B), 15~49 years (group C) and 15-54 years (group D). The consent of parents or guardians was obtained for inclusion of adolescents in the trial. Three vaccine doses were administered. Venepuncture blood was obtained at the time of reading the initial skin test, i.e. immediately before receiving the first vaccination, again at administration of the second and third doses of vaccine, and at 3- and 12-month follow-up when further skin tests for P P D and leishmanial antigen were performed.

Serum samples The number of serum samples for each time point is shown in Table 1. A number of sera from time point 2 were lost in transit from Venezuela to the UK. To permit accurate comparison between timepoints, sera from times 0 through 4 for the same individual were tested on the same plate on the same day for each ELISA. Non-endemic control sera used in the assays were from eight British volunteers (22-35 years old) who had been vaccinated at puberty but had no known exposure to leishmanial antigens. Pools of positive sera for the different ELISAs were as follows: for P P D and BCG from Malawian tuberculosis patients, for MLSA from Malawian lepromatous leprosy patients, and for leishmanial antigen from Indian visceral leishmaniasis patients.

General IgG ELISA method Antigens (mycobacterial and leishmanial as below) were added to 96-well plates (Linbro, ICN Flow) in 100/A Table 1 Number of serum samples for each time point for each of the vaccine groups

Vaccination protocol Full details of the vaccination trial that was carried out in Miranda State near Caracas, Venezuela, have been given previously 13. Briefly, a total of 692 volunteers were screened initially for skin-test reactivity to purified protein derivative of tuberculin (PPD) and leishmanial antigen to obtain 208 double negatives (i.e. ~<7 mm

Time point Group

0

1

2

3

4

A B C D

47 44 44 42

60 41 34 19

20 17 19 8

50 33 30 35

49 28 31 15

Vaccine 1994 Volume 12 Number 15

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Antibody responses to mycobacterial and leishmanial antigens: C.E. Sharpies et al.

V a ¢ ¢ ~ ¢ dose 3

Vaccine d o s e ~

TIME 0

• TIME 1

• TIME 2

.~ TIME 3

... TI3VIE 4

T T T T T Skin test

Skin test

Prebleed

Bleed

Bleed

Bleed

Bleed

Selum

Serum

T cell astei, s

T cell assays

Serum T cell assays

Serum T cell assa,~s

Smum T cell assays

Skin test

Vaccine groups :-

A. B. C. D.

BCG + killed promastigotes BCG alone Killed promastigotes alone Placebo

Figure 1 Diagrammatic representation of the vaccine trial protocol

of carbonate coating buffer (15mM Na2CO3, 35mM NaHCO3, pH9.6) and incubated overnight at 4°C. Coated plates were then washed with phosphate-buffered saline +0.05% Tween 20(137 mM NaC1, 3 mM KC1, 8 mM N a / H P O 4, 1 mM KH2PO4, pH 7.2; PBS-T) and blocked for 1 h at 37°C using 150#l/well of 5% bovine serum albumin (BSA; BDH) in incubation buffer (PBS-T +0.5% BSA). After washing again, the samples were plated in a final volume of 100 #1 incubation buffer and incubated at 37°C for 2 h before washing. Vaccine and control sera were added to the ELISA plates as indicated in each ELISA method by four threefold dilutions, using incubation buffer as a diluent. The positive control pool was titrated on each plate using doubling dilutions from 1 in 20 to 1 in 2560 for BCG and P P D ELISAs, 1 in 50 to 1 in 6400 for the MLSA ELISA and from 1 in 100 to 1 in 12 800 for the leishmanial ELISA. Rabbit anti-human IgG peroxidase conjugate (Dako, Denmark), added to each well at 1 in 1000 dilution in incubation buffer, was incubated for 90min at 37°C. The substrate, 100~l of phosphate-citrate buffer (40mM citrate, 100raM NazHPO4, pH4.5) +0.1% H202 and 2 0 0 # g m l 1 o-phenylenediamine dihydrochloride (OPD; Sigma) was added after washing and developed for 30 min in the dark at room temperature. The reaction was stopped with 50 ~1 1 M H2SO 4 and read in a Dynatech plate reader at 492 rim. Calculating the units in positive pool sera An arbitrary number of units in each positive pool was calculated based on the method described by Hasan et al. z7 A unit was defined as the reciprocal of the dilution

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Vaccine 1994 Volume 12 Number 15

required to raise the absorbance value to twice the background plateau level. The results from the vaccinees and negative controls were calculated as antibody units relative to the positive pool for each dilution tested. The mean of two values taken from the linear part of the pool titration curve was presented as the final value for each sample. Anti-BCG ELISA method ELISA plates were set up as above using a crude preparation of BCG as the coating antigen. BCG (Glaxo) was grown in Middlebrook 7H9 broth (Difco) and the culture tested using Ziehl Neelsen staining for acid-fast bacilli. The crude antigen was prepared by sonication and spinning at 5 0 0 0 0 r e v m i n -1 (Beckman TL-100 Ultracentrifuge) for 30 min at 4°C to remove cell debris. A chequerboard titration was performed against the positive control pool to determine an optimal coating concentration for the ELISA. The positive pool was found to contain 1020 unitsm1-1 anti-BCG lgG antibodies. The positive pool was titrated from I in 20 to 1 in 2560. All the test sera and non-endemic controls were titrated from 1 in 20 to 1 in 720. To eliminate the high background in this ELISA due to the high initial serum concentration, the plates included a 'without antigen' well for all of the samples. Absorbance values for the uncoated wells were subtracted from values for the antigen wells before calculating the number of units. Anti-PPD ELISA method ELISA plates were coated with 10pgml -t PPD (Statens Seruminstitut, Denmark) and included 'without

Antibody responses to mycobacterial and leishmanial antigens: C.E. Sharpies et al.

antigen' wells as in the BCG ELISA because of the high serum concentration. The positive control pool contained 2000 units ml 1 o f a n t i - P P D IgG antibody. All sera were titrated from 1 in 20 to 1 in 2560 for the control pool and to a final dilution of 1 in 540 for the test and non-endemic control sera. Anti-MLSA ELISA method M. leprae soluble antigen (MLSA; batch CD132) provided by Dr D. Rees, NIMR, Mill Hill, U K on behalf of I M M L E P (WHO), was used to coat the plates at 5 p g m l - 1 . Samples were added at the lower starting concentration of 1 in 50 making 'without antigen' wells unnecessary, and titrated to 1 in 1350. The positive pool contained 5000 units and was titrated from 1 in 50 to 1 in 6400 on each ELISA plate.

Anti-LV4 ELISA method L. mexicana (Strain LV4) promastigotes were harvested from mixed logarithmic and stationary-phase liquid cultures, grown in RPMI 1640 (ICN Flow) supplemented with 10% fetal calf serum (Sigma), 100 unitsm1-1 penicillin/streptomycin (Gibco), 2 mM glutamine (Sigma and 1 mM pyruvic acid (Sigma). Promastigotes were washed three times in PBS, sonicated as above, and used without centrifugation to coat the ELISA plates at 5 x 105 parasite equivalents/ml. The plates were washed and

blocked and the samples titrated from 1 in 50 to 1 in 1350. The positive pool contained 40000 units and was titrated from 1 in 100 to 1 in 12 800.

Statistical analyses The non-parametric Wilcoxon paired-sum test was used to compare antibody responses over time as indicated in the results. )~2 analysis was used to determine whether differences in the proportions of responders to non-responders were significant between vaccine groups. RESULTS

IgG responders to mycobacterial and leishmanial antigens The results from the mycobacterial and leishmanial ELISAs are shown as dotplots in Figures 2-5. Tables 2a-5a show the means + s.d. of antibody units in each group. For all assays a wide scatter of antibody units among individuals within each sample group was observed, leading to large standard deviations about the means. As a consequence of this variability, and the fact that the data were not normally distributed, non-parametric tests were used to determine whether significant changes in antibody levels had occurred postvaccination. The Wilcoxon paired-sum test 28 was chosen as the non-parametric equivalent of the paired Student's t test. Each time point was tested for significance by comparison

Group B

Group A

Antibody Level (Ulml; thousands)

Antibody Level (Ulml; thousands)

+ + +

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I g G in v a c c i n e

groups

A-D

Vaccine 1994 Volume 12 Number 15 1405

Antibody responses to mycobacterial and leishmanial antigens: C.E. Sharpies et al. Group B

Group A Antibody Level (Ulml; thousands)

7-

Antibody Level (U/ml; Ihousands)

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Table 3

Anti-PPD IgG levels: (a) Mean 4- standard deviation in each vaccine group for anti-PPD IgG at all time points. (b) Results of Wilcoxon paired-sum tests used to test for significant differences in postvaccination anti-BCG IgG levels compared with prebleed levels. (c) Percentage of responders (n) in each group using a cut-off (mean + 3 s.d. for prebleed over all groups) of 1946 units ml 1 Time points

Time points 0

1

2

3

235_+214 246_+360 196_+189 174_+183

291 _+267 581 +685 2144-316 2754-512

460_+623 546-+889 223_+277 297_+359

Group

4

322_+285 736-+1195 189_+166 229_+340

200_+ 135 745_+1379 240_+573 196_+278

A B C D

T* ~*** NS NS

NS NS NS NS

0 18 3 6

11 2 6 14

T*** T*** NS NS

NS ~** NS NS

2

A B C D

826--+823 585-+395 5824-451 615_+56

680+461 695-+561 597_+372 343_+222

706+_593 581 +342 624+382 344+_167

6 21 0 6

0 15 3 7

A B C D

3

4

946_+878 1162_+285 574_+510 545_+452

661 +474 693_+468 511 +293 263+150

NS t*** NS ~***

~* NS NS NS

NS t*** NS NS

NS NS NS ~**

2 3 0 0

5 0 0 0

10 15 3 0

4 2 0 0

Table 3c

Table 2c

2 2 0 0

(47) (41) (41) (42)

(58) (40) (33) (16)

(18) (17) (18) (7)

*p < 0.05; **p < 0.025; ***p < 0.005; NS, not significant

1406

1

Table3b

Table 2b

A B C D

0

Table 3a

Table 2a

A B C D

4

Dotplots of pre- and postvaccination anti-PPD IgG in vaccine groups A-D

Table 2 Anti-BCG IgG levels. (a) Mean 4- standard deviation in each vaccine group for anti-BCG IgG at all time points. (b) Results of Wilcoxon paired-sum tests used to test for significant differences in postvaccination anti-BCG IgG levels compared with prebleed levels. (c) Percentage of responders (n) in each group using a cut-off (mean + 3 s.d. for prebieed over all groups) of 903 units ml 1

Group

3

2

13me Point

"Rme Point

V a c c i n e 1994 V o l u m e

12 N u m b e r

15

(51) (33) (29) (36)

(50) (27) (31) (14)

A B C D

4 0 0 5

(47) (42) (40) (42)

See footnote to Table 2

(57) (38) (31) (18)

(19) (16) (19) (7)

(50) (33) (30) (35)

(49) (2) (31) (13)

A n t i b o d y r e s p o n s e s to m y c o b a c t e r i a l a n d l e i s h m a n i a l antigens: C.E. S h a r p i e s et al.

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Table 5 Anti-LV4 IgG levels. (a) Mean _+standard deviation in each vaccine group for anti-LV4 IgG at all time points. (b) Results of Wilcoxon paired-sum tests used to test for significant differences in postvaccination anti-BCG IgG levels compared with prebleed levels, (c) Percentage of responders (n) in each group using a cut-off (mean +3 s.d. for prebleed over all groups) of 1282 units m1-1

2

Time points 3

4

Table 4a A 14064-117623624-16032839_+210620004-1473 B 1107___107824174-18952848_+317620754-2213 C 7844-458 1695_+1301 15974-11391012___635 D 9944-693 15704-979 12074-875 998+663 Table 4b A B C D Table 4c A B C D

1`*** 1`*** T*** 1`**

4 5 0 0

(45) (42) (44) (43)

See footnote to Table 2

17 20 6 5

(60) (41) (34) (19)

4

Dotplots of pre- and postvaccination anti-MLSA IgG in vaccine groups A-D

Time points 0

3

Tune Point

Table 4 Anti-MLSA IgG levels: (a) Mean4-standard deviation in each vaccine group for anti-MLSA IgG at all time points. (b) Results of Wilcoxon paired-sum tests used to test for significant differences in postvaccination anti-BCG IgG levels compared with prebleed levels. (c) Percentage of responders (n) in each group using a cut-off (mean +3 s.d. for prebleed over all groups) of 3627 units m1-1

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(20) (15) (19) (8)

(49) (29) (29) (34)

(48) (28) (31) (15)

Group

0

1

2

3

4

Table 5a A 3334-259 11714- 1168 1081 4- 1050 6554-726 B 3704-408 15104-1127 10064-754 4304-455 C 3 6 7 _ + 4 0 4 10564-1459 798_+ 737508_+576 D 2674-193 13704-1592 6824-671 336___253 Table 5b A g

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38 35 11 14

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(16) (17) (18) (7)

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(45) (28) (29) (14)

See footnote to Table 2

Vaccine 1994 V o l u m e 12 N u m b e r 15

1407

Antibody responses to mycobacterial and leishmanial antigens: C.E. Sharpies et al. Group A 7-

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with the prevaccination antibody values. Only samples for which there were paired values could be used in this statistical analysis. Antibody responses to BCG

Results from the BCG ELISA are shown as dotplots in Figure2, means _+s.d. in Table2a, and Wilcoxon paired-sum test analysis in Table2b. Anti-BCG IgG antibody levels increased significantly only in those vaccine groups that had received BCG, with the largest increase observed in group B that received BCG alone. The absence of Leishmania in the vaccine inoculum resulted in an increased and more sustained humeral response to BCG. At time 4, 1 year after the initial vaccination, the antibody levels were still significantly higher than the prebleed level. The small number of samples at time 2 most probably contributed to the lack of significance shown at this time point. Groups C and D, who did not receive any BCG vaccination, showed no significant increase in anti-BCG IgG levels postvaccination. However, their mean antibody levels remained higher at ~200 unitsm1-1 than that for the BCG-vaccinated non-endemic controls (83+_33 units ml- 1, n = 8). Using the mean of all the vaccine groups at the prebleed time point + 3 standard deviations (mean = 207 + 232) as a cut-off (903 units ml-l), responder versus non-

1408

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Vaccine 1994 Volume 12 Number 15

Point

a n t i - L V 4 I g G in v a c c i n e g r o u p s A - D

responder status for individuals at the postvaccination time points was determined (Table 2c). A high proportion of non-responders was observed in all groups. However, group B, which was vaccinated with BCG alone, had a significantly higher proportion of responders than other groups at times 1 ( Z 2 = 13.34; degrees of freedom (d.f.)= 3; 0.001 < p < 0.01), 3 (X2 = 10.76; d.f. = 3; 0.01 < p < 0.05) and 4 (Z2=8.57; d.f.=3; 0.01
Results from the P P D IgG ELISA are shown as dotplots in Figure3, means+s.d, in Table3a, and Wilcoxon paired-sum test analysis in Table 3b. As with BCG antigen, enhanced postvaccination levels of anti-PPD IgG occur only in group B (BCG alone). The statistics show that the presence of killed promastigotes in the inoculum of group A abrogated any potential antibody response to PPD. At time 2, this group in fact shows a significant decrease in antibody units. Curiously,

Antibody responses to mycobacterial and leishmanial antigens." C.E. Sharpies et al. the placebo group also showed a significant decrease at time 1 and time 4 after vaccination. Group C, which did not receive any BCG, did not alter its mean antibody level throughout the trial. As for the BCG ELISA, responses in non-endemic controls had a much lower level of anti-PPD units in their sera, giving a mean of 34 + 23 (n = 7) units m l - 1. Using a prevaccination cut-off (1946 units ml- 1) of mean + 3 standard deviations, the percentages of responders in the vaccine groups were determined (Table 3c). The data show higher proportions of responders in groups A and B at time 3 compared with the non-BCG vaccinated groups C and D, although this fails to attain statistical significance ()~2= 6.9; d.f. = 3; 0.05 < p <0.1). Antibody responses to MLSA Responses to MLSA are shown as dotplots in Figure 4, means + s.d. in Table 4a, and Wilcoxon paired-sum tests in Table 4b. For this assay, all groups exhibited enhanced antibody production at time 1 compared with the prebleed level. Groups A and B, who both received BCG in their vaccination, showed significant increases in units of anti-MLSA IgG. This increase was highest at the time of the second and third vaccinations and had waned by the 1-year follow-up. The addition of heat-killed parasites in the vaccine inoculum of group A had no effect on the antibody levels compared with group B. Group C (killed promastigotes alone) also showed a significant (p < 0.005) increase after vaccination and this was present at the final review of the vaccinees. The mean _+s.d. values (Table 4a) reflect the wide range of responses found in each group. The prevaccination responses are similar to those seen in non-endemic controls (886_+ 187, n = 8). Responder/nonresponder status was determined as before (Table4c; cut-off 3627 unitsml-1). A high proportion of nonresponders was observed in all vaccine groups. However, a significantly higher proportion of responders to non-responders occurs over times 1, 2 and 3 in groups A and B which received BCG vaccination (;(2= 12.56; d.f.=6; p=0.05). Using this cut-off, none of the non-endemic control individuals was found to be a responder. Antibody responses to LV4 Anti-LV4 antibody levels are shown as dotplots in

Figure 5, means__, s.d. in Table5a, and Wilcoxon paired-sum tests in Table 5b. Time 0 levels of anti-LV4 IgG were marginally higher than the mean for the eight non-endemic volunteers (256_+ 52 units m l - 1). With this ELISA, all of the vaccination groups increased their anti-LV4 antibody level through the vaccination schedule according to the Wilcoxon rank-sum test (Table5b). Plotting the means against time (and month of collection) revealed a pattern in all groups (including the placebo) indicative of seasonal variation independent of vaccine status (Figure6a). Alternatively, the groups may be responding to the promastigote antigen present in the skin-test antigen administered before vaccination and again at time 3. If this is the case, a parallel influence of the mycobacterial skin-test antigen on antibody responses to mycobacterial antigens (e.g. MLSA, Figure 6b) did not occur, except perhaps in the groups (C and D) not receiving BCG. The percentage of anti-LV4 responders (cut-off 1282 unitsm1-1) at each time point (Table5c) followed the same trend seen in Figure6. A similar

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number of responders was found in each group and responders were at the highest frequency at times 1 and 2, and lowest at time 3. Groups A and B, who had received BCG, had a higher proportion of responders to non-responders than with groups C and D, raising the possibility that BCG boosted the antibody response to endemic or skin-test administered leishmanial infection/antigen. This did not, however, attain statistical significance (Z2= 13.56; d.f.=9; NS). It should be noted too that the anti-LV4 antibody responders identified using the criterion of mean + 3 standard deviations above prevaccination levels nevertheless produced antibody levels orders of magnitude lower than that (40000 units ml- 1) observed in the positive visceral leishmaniasis patient control pool.

V a c c i n e 1994 V o l u m e 12 N u m b e r 15

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Antibody responses to mycobacterial and leishmanial antigens: C.E. Sharpies et al.

DISCUSSION One of the central immunological features of both leishmanial and mycobacterial infections in humans is that severe forms of disease, for example diffuse or mucocutaneous leishmaniasis and lepromatous leprosy, are associated with extremely high titres of circulating IgG which play no protective role. Recent studies have confirmed that individuals presenting with these forms of disease make an inappropriate T-cell response with a bias towards TH2-associated cytokines detected both in the lesions 2°'22 and in peripheral blood responses 2~. In contrast, self-healing forms of disease are associated with production of macrophage-activating interferon-7 and a T H l - t y p e response TM 22.One of the major concerns in vaccination strategies based on whole crude organisms has been the possibility that the response to vaccination for some individuals in the population might also be biased towards a TH2-associated response. This is a particular concern in the context of cutaneous leishmaniasis where murine models of vaccination based on whole crude promastigote preparations have demonstrated that subcutaneous inoculation of the vaccine leads to disease exacerbation rather than protection 29 3~. Results presented here demonstrate that subcutaneous vaccination with crude leishmanial promastigotes with or without BCG did not itself lead to high titres of antileishmanial or antimycobacterial IgG. Indeed, when prevaccination lgG levels were used to determine a negative cut-off, very few vaccines converted to responder status, and those who did would be classified as 'low-responder' status compared with titres observed 23 25.27,32.33 in severe forms of disease. Hence, although there was evidence for a background endemic response to both leishmanial and mycobacterial antigens, there was no evidence that vaccination per se led to a potentially disease exacerbatory level of TH2-associated antibody response. Indeed, in the absence of antibody subclass data, it is impossible to determine whether the low levels of antibody observed in vaccinated individuals were regulated by TH2 or T H 1 pathways. Taken together with our earlier report that a high proportion of vaccinees, particularly in the BCG plus killed promastigotes group ( > 90%), converted for T-cell responder phenotypes (skin test: proliferative response; interferon-7 production) to leishmanial antigens, our results indicate that this vaccine is potentially protective for the majority of vaccinees. This justifies a level of confidence in extending vaccination trials utilizing whole killed organisms to test vaccine efficacy in preventing disease. There were, however, some interesting aspects of the antibody profiles to the different mycobacterial antigens and to leishmanial antigen which bear further discussion in relation to previous studies. In our study the prevaccination background levels of response to mycobacterial antigens were higher in the vaccinees than in non-endemic controls, despite that fact that the latter group had been BCG-vaccinated at puberty. This is presumably due to repeated exposure to antigen in the TB/leprosy endemic regions in which this Venezuelan study was undertaken. Previous studies of antibody responses in tuberculosis 32'34'3s patients have also reported higher background responses in healthy endemic compared with non-endemic controls, but with no 3'~'36 3s or only low or transient 39'4° evidence for enhanced antimycobacterial (TB, BCG or PPD) IgG in BCG-vaccinated versus non-vaccinated groups. In our 1410

Vaccine 1994 Volume 12 Number 15

study there was a concern, however, that in selecting vaccine volunteers on the basis of negative skin-test responses to P P D and leishmanial antigen we may have biased towards a volunteer group with high antibody and low T H l - t y p e response potential. Since we did not collect samples from the skin-test positive 'reject" population, we do not know whether our group had a higher background antibody response than skin-test positive individuals. In fact, other studies have reported the contrary observation that IgG responses to M. tulwrculosis antigen was higher in individuals who were tuberculin-positive but did not have active disease ~''. Importantly, results presented here and in our previous report ~3 demonstrate that our vaccine group selected on the basis of skin-test negativity neither remained solidly non-responders in terms of cell-mediated immunity nor became uniformly high antibody responders postvaccination. It is clear, nevertheless, that the response of individuals could be biased either towards a T H I associated, interferon-7-producing, cell-mediated immune response, or towards a TH2-associated antibody response. The factors (genetic and environmental) regulating this bias could be important in determining vaccination success or failure at the individual level. This may be relevant to BCG vaccination trials against both leprosy and tuberculosis, and merits further analysis. For antibody responses to leishmanial antigen, similarly enhanced background IgG titres were observed compared with non-endemic controls. In previous studies, anti-leishmanial lgG found in patients with active localized cutaneous leishmaniasis caused by Leishnumia braziliensLs' have been shown to be higher on average than healthy skin-test positive or skin-test negative endemic individuals 23 25.~, although some patient levels fall within the range of endemic controls 4~. Again, by selecting skin-test negative individuals into the vaccine trial, the sample may have been biased towards high antibody/low TH 1-type responders, even though the lgG titres observed were much lower than those associated with active localized or severe diffuse or mucocutaneous disease23 25.4~. What was interesting, however, was that antileishmanial IgG levels increased in all vaccine groups independently of whether leishmania[ antigen was included in the vaccination inoculum received. Although this could have been due to response to the skin-test antigen, the pattern of response observed in all vaccine groups was more suggestive of a seasonal cycle in response to endemic exposure. Since Leishmania is a vector-borne parasite, seasonal variation in exposure would be expected compared with the continuous exposure associated with mycobacterial disease such as TB/leprosy. Seasonal patterns of disease caused by L. hraziliensis were reported in Brazil between 1979 and 1981, with most cases occurring between April and July, the months with the highest rainfall "~2. This matches precisely the increase in antibody levels observed between April and June in our study. Unlike the responses to mycobacterial antigen in vaccinees receiving BCG, there is no evidence of an enhanced postvaccination lgG response in the groups receiving leishmanial antigen. This contrasts with a previous South American vaccination trial 43 in which increases in anti-leishmanial antibodies detected by ELISA were observed in individuals immunized with killed promastigotes from a mixture of five or six New World Leishmania strains with or without Corynebacterium pan'urn as an adjuvant. Interestingly,

A n t i b o d y r e s p o n s e s to m y c o b a c t e r i a l a n d l e i s h m a n i a l antigens: C.E. Sharpies et al.

the IgG antileishmanial antibody response to crude leishmanial antigen was highest in the two groups that received C. parvum, suggesting again that addition of a non-specific stimulus at the time of immunization might bias towards a TH2-type response. In the South American study antibodies were also evident in the placebo group. Nascimento and co-workers 43 attributed this to the skin-test antigen, although it could presumably have been due to endemic exposure. Subsequent studies in Brazil 44 have indicated that antibody responses are not induced by skin-test antigen alone, although administration of skin-test antigen will enhance antibody responses in previously sensitized individuals. One interesting observation made here was the opposing effects of killed leishmanial antigen on anti-BCG responses versus BCG on anti-leishmanial antibody levels. Vaccinees receiving BCG alone showed enhanced responses to mycobacterial antigens. For anti-MLSA responses, parallel results were obtained in the groups receiving BCG alone or with killed promastigotes. For anti-BCG and anti-PPD responses, however, the BCG + killed promastigotes group showed a reduced response compared with the BCG alone group. Hence, the presence of leishmanial antigen appeared to suppress antibody responses to M. bovis/M, tuberculosisrelated antigens. In contrast, administration of BCG (with/without leishmanial antigen) led to an increased number of antileishmanial |gG responders. Although this appeared to be an enhanced endemic- or skin-test antigen-associated rather than vaccine-related response, it does suggest that administration of BCG could lead to a potentially exacerbatory TH2-associated antibody response in a proportion of individuals, and also that there might be an inverse relationship in such individuals between their TH1- and TH2-associated responses and polymorphism at the genes that control these responses. In a further report (Sharpies, to be submitted) T-cell and antibody responses of individual vaccines are examined specifically in relation to polymorphism at their MHC class II (HLA-DR) and class III (TNF-/~) genes.

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ACKNOWLEDGEMENTS Financial support for this work was provided by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases Projects (Numbers 880178 and 930102), by the Wellcome Trust, and by the British Medical Research Council. The authors are grateful to Drs Pedro Luis Castellanos, Santina Formica and Arcady Rodas, as well as to other members of the field team, for their dedication to the vaccination protocol; and to Dinorah Trujillo for processing serum samples.

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REFERENCES 23 1 Greenblatt, C.L. Cutaneous leishmaniasis: the prospects for a killed vaccine. Parasitol. Today 1988, 4, 53 2 Salles Gomes, L. A intra-dermo-reaccao de Montenegro na leishmaniose e outras pesquizas affins. BrasiI-Medico 1939, 53, 1079 3 Pessoa, S.B. and Pestana, B.R. Ensaio sobre a vacinacao preventiva na leishmaniose tegumentat americana, corn germans mortas. Rev. Biol. Hig. 1940, 10, 112 4 Mayrink, W., Magalhaes, P.A., Dias, M., da Costa, C.A., Melo, M.N. and Oliveira Lima, A. Responses to Montenegro antigen after immunization with killed Leishmania promastigotes. Trans. R. Soc. Trop. Med. Hyg. 1988, 72, 676

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Mayrink, W., da Costa, C.A., Magalhaes, P.A., Melo, M.N., Dias, M., Oliveira Lima, M.S. et al. A field trial of a vaccine against American dermal leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 1979, 73,385 Mayrink, W., Williams, P., da Costa, C.A., Magalhaes, MN., Melo, M.N., Dias, M. et al. An experimental vaccine against American dermal leishmaniasis: experience in the state of Espirito Santo, Brazil. Ann. Trop. Med. Parasitol. 1985, 79, 259 Antunes, C.M.F., Mayrink, W., Magalhaes, P.A., da Costa, C.A., Melo, MN., Dias, M. et al. Controlled field trials of a vaccine against New World cutaneous leishmaniasis. Int. J. Epidemiol. 1986,15, 572 Mayrink, W., Antunes, C.M.F., da Costa, C.A., Melo, M.N., Dias, M., Michalick, M.S. et al. Further trials of a vaccine against American cutaneous leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 1986, 88, 1001 Howard, J.G., Nicklin, S., Hale, C. and Liew, F.Y. Prophylactic immunization against experimental leishmaniasis. I. Protection induced in mice genetically vulnerable to fatal Leishmania tropica infection. J. Immunol. 1982, 1%)9,2206 Scott, P., Pearce, E., Natovitz, P. and Sher, A. Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes. J. Immunol. 1987, 139, 221 Russell, D.G. and Alexander, J. Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes. J. Immunol. 1988, 140, 1274 Handman, E. and Mitchell, G.F. Immunization with Leishmania receptor for macrophages protects mice against cutaneous leishmaniasis. Proc. Natl Acad. Sci. USA 1985, 82, 5910 Castes, M., Blackwell, J., Trujillo, D., Formica, S., Cabrera, M., Zorilla, G. et al. Immune response in healthy volunteers vaccinated with BCG plus killed leishmanial promastigotes: Skin test reactivity, T ceil proliferation and interferon-T production. Vaccine 1994, in press Convit, J., Rondon, A., Ulrich, M., Bloom, B., Castellanos, P.L., Pinardi, ME. et al. Immunotherapy versus chemotherapy in localized cutaneous leishmaniasis. Lancet 1987, 8530, 401 Convit, J., Castellanos, P.L., Ulrich, M., Castes, M., Rondon, A., Pinardi, M.E. et al. Immunotherapy of localized, intermediate and diffuse forms of American cutaneous leishmaniasis. J. Infect. Dis. 1989, 160, 104 Heinzel, F.P., Sadick, M.D., Holaday, B.J., Coffman, R.L. and Locksley, RM. Reciprocal expression of interferon- 7 or interleukin 4 during the resolution or progression of murine leishmaniasis. J. Exp. Med. 1989, 169, 59 Scott, P., Natovitz., P., Coffman, R.L., Pearce, E. and Sher, A. Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens. J. Exp. Med. 1988, 168, 675 Rada, E, Trujillo, P.L, Castellanos, P.L. and Convit, J. Gamma-interferon producton induced by antigens in patients with leprosy and American cutaneous leishmaniasis. Am. J Trop. Med. Hyg. 1987, 37, 520 Castes, M., Cabrera, M., Trujillo, D. and Convit, J. T cell populations, expression of IL-2 receptor and production of IL-2 and interferon- 7 in human American cutaneous leishmaniasis. J. Clin. Microbiol. 1988, 26, 1207 Caceras-Dittman, G., Tapia, F.J., Sanchez, M.A., Yamamura, M., Uyemura, K., Modlin, R.L. et al. Determination of the cytokine profile in American cutaneous leishmaniasis using the polymerase chain reaction. Clin. Exp. Immunol. 1993, 91,500 Kemp, M., Hey, A.S., Kurtzhaels, J.A.L., Christersen, C.B.V., Gaafar, A., Mustafa, M.D. et al. Dichotomy of the human T ceil response to Leishmania antigens. I. Thl-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis. Clin, Exp. Immunol. 1994, 96, 410 Pirmez, C., Yamamura, M., Uyemura, K., Paes-Oliveira, M. and Modlin, R.L. Cytokine patterns in the pathogenesis of human leishmaniasis. J. Clin. Invest. 1993, 91, 1380 Convit, J. and Pinardi, M.E. Applying the indirect immunofluorescence test to the study of American cutaneous leishmaniasis. Dermatol. Int. 1969, 8, 17 Convit, J. and Pinardi, M.E. Cutaneous leishmaniasis. The clinical and immunopathological spectrum in South America. CIBA Found. Symp. 1974, 20, 159 Mendon(~a, S.C.F., Souza, W.J., Nunes, M.P., Marzochi, M.C. and Coutinho, S.G. Indirect immunofluorescence test in New World leishmaniasis: serological and clinical relationship. Mem. Inst. Oswaldo Cruz 1988, 83, 347-355 Britton, W.J. Heilqvist, L., Basten, A. and Inglis, A.S. Immunoreactivity of a 70KD protein purified from Mycobacterium bovis

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bacillus Calmette-Guerin by monoclonal antibody affinity chromatography. J. Exp. Med. 1986, 164, 695 Hasan, R., Dockrell, H.M., Chiang, T. and Hussain, R. Quantitative antibody ELISA for leprosy. Int. J. Lepr. Other Mycobact. Dis. 1989, 57, 766 Bailey, N.T.J. Non-parametric and distribution free tests. In: Statistical Methods in Biology (Ed. Bailey, NT.J.) Hodder and Stoughton, London, 1985 Liew, F.Y., Hale, C. and Howard, J.G. Prophylactic immunization against experimental leishmaniasis. IV. Subcutaneous immunization prevents the induction of protective immunity against fatal Leishmania major infection. J. Immunol. 1985, 135, 2095 Liew, F.Y., Singleton, A., Cillari, E. and Howard, J.G. Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection. J. Immunol. 1985, 135, 2102 Liew, F.Y., Hodson, K. and Lelchuk, R. Prophylactic immunization against experimental leishmaniasis. Vl. Comparison of protective and disease-promoting T cells. J. Immunol. 1987, 139, 3112 Nassau, E., Parson, E.R. and Johnson, G.D. The detection of antibodies to Mycobacterium tuberculosis by microplate enzymelinked immunosorbent assay (ELISA). Tubercle 1976, 57, 67-70 Grange, J,M., Gibson, J., Nassau, E. and Kardjito,T. Enzyme-linked immunosorbent assay (ELISA): a study of antibodies to Mycobacterium tuberculosis in the IgG, IgA and IgM classes in tuberculosis, sarcoidosis and Crohn's disease. Tubercle 1980,61,145 Dubina, J., Dobah, S, Danes, L., Kopecky, K., Kubin, M., Prochazka,B. and Wisingerova, E. Screening of serum antibodies against Mycobacterium tuberculosis by enzyme-linked immunosorbent assay (ELISA) in health population. J. Hyg. Epidemiol. Microbiol. Immunol. 1989, 1, 83 Krambovitis, E. Detection of antibodies to Mycobacterium tuberculosis plasma membrane antigen by enzyme-linked immunosorbent assay. J. Med. Microbiol. 1986, 21,257 Kardjito, T., Handoyo, I. and Grange, J.M. Diagnosis of active

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tuberculosis by immunological methods. 1. The effect of tuberculin reactivity and previous BCG vaccination on the antibody levels determined by ELISA. Tubercle 1982, 6,3, 269 Kalish, S.B., Radin, R.C., Phair, J.P., Levitz, D., Zeiss, C.R. and Metzger, E. Use of enzyme-linked immunosorbent assay technique in the differential diagnosis of active pulmonary tuberculosis in humans. J. Infect. Dis. 1983, 147, 523 Balestrino, E.A., Daniel, T.M., de Latini, M.D.S., Latini, O.A., Ma, Y. and Scocozza, J.B. Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbant assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative. Bull. World Health Organ. 1984, 62, 755 Rosen, E.U. The diagnostic value of an enzyme-linked immune sorbant assay using adsorbed mycobacterial sonicates in children. Tubercle 1990, 71, 127 Turneer, M., Van Vooren, J.P., Nyabenda, J., Legros, F., Lecomte, A., Thiriaux, J. et al. The humoral immune response after BCG vaccination in humans: consequences for the serodiagnosis of tuberculosis. Eur. Respir. J. 1988, 1,589 Masuda, A., Nascimento, S.F., Guerra, C.S., Paranhos, G. and Ferreira, A.W. Analysis of the specificity of human antibodies to antigens of Leishmania braziliensis. Bev. Inst. Med. Trop. Sao Paulo 1989, 31,228 Jones, T.C., Johnson, W.D., Barretto, A.C., Lago, E., Badaro, R., Cerf, B. et al. Epidemiology of American cutaneous leishmaniasis due to Leishmania braziliensis braziliensis. J. Infect. Dis. 1987, 156, 73 Nascimento, E., Mayrink, W., da Costa, C.A., Michalick, M.S.M., Melo, M.N., Barros, G.C. et al. Vaccination of humans against cutaneous leishmaniasis: cellular and humoral immune responses. Infect. Immun. 1990, 58, 2198 Nascimento, M.D.S.B., Alcantara-Neves, NM., Muniz, M.E.B., Nunes, S.F., Paranhos, M. and Pontes de Carvalho, L.C. Induction of the immune response to Leishmania by Montenegro's skin test. Trans. R. Soc. Trop. Med. Hyg. 1993, 87, 91