Delayed-type hypersensitivity in volunteers immunized with a synthetic multi-antigen peptide vaccine (PfCS-MAP1NYU) against Plasmodium falciparum sporozoites

Vaccine 20 (2002) 1853–1861

Delayed-type hypersensitivity in volunteers immunized with a synthetic multi-antigen peptide vaccine (PfCS-MAP1NYU) against Plasmodium falciparum sporozoites James G. Kublin a , Mark H. Lowitt b , Robert G. Hamilton c , Giane A. Oliveira d , Elizabeth H. Nardin d , Ruth S. Nussenzweig d , Barbara J. Schmeckpeper e , Carter L. Diggs f , Sacared A. Bodison g , Robert Edelman a,∗ a

c

Department of Medicine and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, USA b Department of Dermatology, University of Maryland School of Medicine, Baltimore, MD, USA Reference Laboratory for Dermatology, Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD, USA d Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY, USA e Immunogenics Laboratories, Johns Hopkins University School of Medicine, Baltimore, MD, USA f Malaria Vaccine Development Program, Agency for International Development, Washington, DC, USA g University Health Center, University of Maryland, College Park, MD, USA Received 8 June 2001; received in revised form 30 November 2001; accepted 3 December 2001

Abstract During the testing of the safety and immunogenicity of an adjuvanted, synthetic Plasmodium falciparum CS multiple antigen peptide (MAP) vaccine, we investigated the potential for using cutaneous delayed-type hypersensitivity (DTH) reactions as a correlate of immune response. We evaluated 27 of our volunteers for DTH reactions to intradermal inoculation (0.02 ml) of several concentrations of the MAP vaccine and adjuvant control solutions. Induration was measured 2 days after skin tests were applied. Nine of 14 vaccinees (64%) with serum, high-titered anti-MAP antibody developed positive DTH (≥5 mm induration), that first appeared by 29 days after immunization and persisted for at least 3–6 months after 1–2 more immunizations. In contrast, DTH responses were negative in eight of eight vaccinees with no or low antibody titers, and in five of five non-immunized volunteers. Biopsies of positive DTH skin test sites were histologically compatible with a DTH reaction. We conclude that the presence of T cell functional activity reflected by a positive DTH skin test response to the MAP antigen serves as another marker for vaccine immunogenicity. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Malaria vaccine; Cutaneous delayed-type hypersensitivity; Phase I vaccine trial

1. Introduction Morbidity and mortality associated with malaria infection are major public health problems throughout much of the world. Obstacles to the control of malaria, such as drug resistance and expanding vector populations, highlight the need for a malaria vaccine. Several malaria antigen vaccine candidates are being developed and means to assess vaccineinduced immune response and efficacy in humans are needed. Naturally-acquired and vaccine-induced immunity lead to protection through several immunologic mechanisms. The in vitro assays available to measure these mechanisms provide indirect estimates of biological activity (e.g. in vitro reduction in parasite numbers or ∗ Corresponding author. Tel.: +1-410-706-8443; fax: +1-410-706-6205. E-mail address: [email protected] (R. Edelman).

growth, gamma-interferon release from T cells in vitro, and antibody to sporozoite surface antigen), but in vivo markers of immunogenicity would be of great value [1,2]. Delayed-type hypersensitivity (DTH) skin testing was used as a diagnostic tool for malaria over 70 years ago [3]. DTH is used most commonly to detect exposure to a sensitizing dose of antigen through vaccination or infection, as in the use of the purified protein derivative (PPD) in testing for Mycobacterium tuberculosis infection. DTH skin testing is also used to assess immunocompetence in response to a battery of common recall antigens, to assess prognosis in HIV infection by predicting AIDS progression, and to examine in vivo the cell-mediated response to vaccines and infection [4–6]. DTH has more recently been employed to map epitopes of candidate HIV vaccine antigens [7]. The synthetic Plasmodium falciparum multiple antigen peptide (MAP) vaccine, which contains minimal B and T

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cell epitopes from the repeat regions of the circumsporozoite (CS) protein [8], was developed when limitations of the peptide–protein conjugate vaccine were recognized following Phase I/II trials in the late 1980s [9,10]. MAP vaccines containing T and/or B cell epitopes of malaria proteins elicited high levels of antibody mediated protection in mice and monkeys [11,12], as well as CD4+ T cell ␥-interferon (␥-IFN) dependent protective immunity [13,14]. Subsequently, a MAPs vaccine designated (T1B)4 MAPs, which was constructed with B and T cell epitopes from the CS repeat region, demonstrated a significant anamnestic reponse when administered to P. falciparum sporozoite-primed mice and monkeys [15]. The (T1B)4 MAP high responder phenotype correlated with murine H-2b haplotype [16], and limited genetic restriction was also observed in vitro with certain human HLA class II molecules [17]. In preclinical studies, three HLA class II molecules, DRB1∗ 0401, DRB1∗ 1101, and DQB1∗ 0603, were termed “responder” genotype, based on the ability to bind in vitro the T1 peptide, which is contained in the (T1B) MAP vaccine. During a prospective, open-label Phase I clinical trial of the (T1B)4 MAP vaccine, volunteers were selected based on their class II genotypes to represent responder HLA; volunteers with random HLA haplotypes were also included in the study [18]. We performed intradermal skin testing to correlate DTH with the humoral response to the MAP vaccine in a subset of these vaccinees.

2. Materials and method 2.1. Volunteer subjects The selection of healthy adult volunteers, the vaccine and adjuvant formulations, and the study design have been previously described [18]. Informed consent was obtained from all volunteers, according to the human experimentation guidelines of the US Department of Health and Human Services. The Institutional Review Boards of the University of Maryland, College Park and the University of Maryland, Baltimore, approved the study. In brief, 88 healthy adult volunteers (18–45 years old) were screened for HLA class II haplotypes using PCR amplification of DNA extracted from peripheral blood lymphocytes (Immunogenetics Laboratory, Johns Hopkins Medical Institutions). The PCR products were dot-blotted to membranes and hybridized with radio-labeled, sequence specific oligonucleodide probes using a standard typing scheme of the 11th and 12th International Histocompatibility Workshops [19]. Cohort A was identified from 51 screened volunteers recruited at the University of Maryland, College Park campus, of whom 32 were immunized. Cohort B was selected from 37 screened recruits from the Baltimore, MD community, 14 (38%) of whom had HLA responder genotypes. Seven of these 14 were suitable for the study and immu-

nized. Exclusion criteria for the study included a history of malaria or malaria vaccination; atopy; cardiovascular, hepatic, or renal disease; substance abuse during the past year; iatrogenic immunosuppression; splenectomy; abnormal serum immunoglobulin levels, electrolytes, or renal or hepatic chemistries; or positive serological tests for Hepatitis B or C viruses, or the human immunodeficiency virus. The volunteers were expected to be available for 12 months after initiation of the study to assure complete follow-up. Informed consent was obtained from all volunteers. All volunteers were required to score 70% or better on a 20 question, multi-choice exam given to test their knowledge of the purpose, procedures, benefits and risks of the study. 2.2. Vaccine and adjuvant formulations The PfCS-MAP1NYU [(T1B)4 MAP)] construct (hereafter referred to as MAP) is comprised of four identical linear peptides each of which contains the immunodominant B-cell epitope [NANPNANPNANP] and a T-cell epitope (T1) [DPNANPNVDPNANPNV] [20]. These are linked through a matrix of three lysines to an (acetamido)-cysteinyl-alanyl chain. The MAP was synthesized under GMP standard Merrifield solid phase peptide synthesis by Peninsula Laboratories, Inc. (Belmont, CA 94002). Purity and composition of the final MAP construct was assessed by HPLC and amino acid analysis. The MAP was adsorbed to aluminum hydroxide (alum) (Reheis, Inc., Berkeley Heights, NJ) at the Walter Reed Army Institute of Research. The final concentration of the vaccine in each vial was 2.5 mg MAP and 3.1 mg aluminum per ml; 70% of the antigen was adsorbed to aluminum hydroxide. QS-21 in PBS (Aquila Biopharmaceuticals, Inc., Framingham, MA) was added as a co-adjuvant. QS-21 is a saponin derivative that enhances the immunogenicity of veterinary vaccines and several human vaccines [21–23]. Preclinical studies showed that QS-21 significantly increased the antibody response of mice and Aotus monkeys to the MAP/alum vaccine [24]. The QS-21 and MAP/alum were mixed immediately before immunization to obtain the desired concentrations of vaccine and adjuvants. 2.3. Study design In cohort A, the study was an open-label trial to test increasing concentrations of the MAP antigen adsorbed to alum or adsorbed to alum and mixed with QS-21. The purpose was to assess vaccine safety and to determine whether the T1 epitope, when incorporated into a MAP vaccine, could elicit CD4+ T helper cell responses in volunteers expressing HLA responder alleles. We examined the dose response in 32 individuals to 500 or 1000 ␮g of the MAP/alum vaccine alone or in combination with 50 or 100 ␮g of QS-21 [18]. Each group of eight adults was immunized with 0.6 ml of one of the four vaccine formulations injected subcutaneously (s.c.) into the upper arm;

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injections were administered three times into alternate arms at 0 and 28 days, and 2–8 months. The study design for cohort B was developed to re-assess the safety and immunogenicity of the MAP vaccine, to confirm genetic restriction and to assess protective efficacy via challenge with malaria-infected mosquito bites. Seven individuals with HLA responder alleles were selected for the study. Because urticaria was observed in two volunteers from cohort A, skin testing for immediate-type hypersensitivity (ITH) was integrated into the study design to assess for the potential development of allergy to the vaccine (Edelman et al., submitted). Skin testing also allowed us to assess DTH using the same skin test reagents, and to correlate the cutaneous DTH response with in vitro measurements of the immune response. 2.4. Clinical study procedures In cohort A, Groups 1 and 2 were immunized on days 0, 28 and 56 with 500 ␮g MAP/alum and 500 ␮g MAP/alum + 50 ␮g QS-21, respectively. Group 3 volunteers were immunized on days 0, 28 and 56 with 1000 ␮g MAP/alum+50 ␮g QS-21. Group 4/5 was immunized on days 0, 28, and 237 with 1000 ␮g MAP/alum mixed with 100 ␮g QS-21 The administration of the third dose of vaccine was delayed until day 237 in Group 4/5 following the development of urticaria in two of eight volunteers in the previous test group, Group 3. At this time, seven months after the second vaccination, Group 4/5 volunteers were negative by ITH skin test and antibody titers had decreased to <1:1000. The third dose of vaccine was administered to Group 4/5 volunteers without any adverse reactions (Edelman et al., submitted). Participants in cohort B were immunized with two injections of a MAP vaccine formulation consisting of 500 ␮g MAP adsorbed to 630 ␮g alum and mixed with 50 ␮g QS-21. After negative allergy skin tests to the MAP vaccine on day 27, they were safely immunized a second time on day 28. Allergy skin tests were repeated 14 and 83 days after the second immunization, but because of newly-developed allergy skin test reactivity, the third (booster) injection of MAP vaccine was postponed and eventually canceled (Edelman et al., submitted). After each vaccine injection volunteers were observed for local and systemic reactions for the first 60 min and at regularly scheduled clinic visits at 24 and 48 h and at 7, 14, and 28 days after immunization. In addition, any interval history recorded by the volunteer was confirmed at each clinic visit. The vaccinees’ assessment of the injection and details of the systemic signs and symptoms, clinical laboratory tests (Edelman et al., submitted), and serology and cellular responses of the vaccinees [18]are reported elsewhere. Blood samples were collected from each volunteer for antibody titers on the day of immunization, 2 weeks after each immunization, and at 3, 5, 8 and 12 months after the third immunization of cohort A or after the second immunization of cohort B.

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2.5. Serological assays Peptide specific antibodies were measured by ELISA using HRP-labelled antibodies specific for Fc of human IgM or IgG (Cappel, West Chester, PA). ELISA were performed using 96-well plates coated with 100 ␮l of 1 ␮g/ml of MAP per well, as described [18]. Indirect immunofluorescent antibody (IFA) assays employed air-dried P. falciparum sporozoites incubated with two-fold dilutions of sera, followed by FITC-labelled anti-human IgG or IgM (Kierkegaard & Perry, Gaithersburg, MD) diluted in PBS/0.04% Evans blue [18]. 2.6. Delayed-type hypersensitivity (DTH) skin tests Intradermal skin tests (ID-ST) were applied using MAP/alum/QS-21 vaccine dilutions and alum/QS-21 control dilutions. Test agents were sequentially injected in duplicate, at 15 min intervals, starting with the highest dilutions. For ID-STs, we employed a standard method [25], and injected 0.02 ml of the MAP vaccine or adjuvant solutions intradermally on the volar surface of the forearm using a 1.0 ml tuberculin syringe and 27 gauge needle. In cohort A, Group 3 and Group 4/5 (8 volunteers each) were tested once, 7 months after the third and second vaccinations, respectively. In cohort B, volunteers were tested three times to further characterize the temporal development of the DTH response and to correlate DTH with the antibody response. Skin tests of cohort B were applied 1 day before and 14 and 83 days after the second vaccination. The immediate wheal and flare reactions (ITH responses) at these sites, recorded at 15 min, are reported elsewhere (Edelman et al., submitted). The diameter of induration (and erythema) at the test sites were measured at right angles using a millimeter ruler and ball point pen [26]. The DTH responses were measured without knowledge of the HLA type and antibody status of the volunteer. Cohort A measurements were on day 2 and/or day 7, and cohort B measurements were made on day 2, although if negative or indeterminate, remeasured on day 7. Positive DTH skin test reactions were defined as ≥5 mm induration to the MAP vaccine reagent and ≤1 mm induration to the adjuvant control reagent at the sites where 0.02 ml of ID-ST solutions had been injected. Negative DTH skin test reactions were defined as ≤1 mm induration to the MAP vaccine reagent and ≤1 mm induration to the adjuvant control reagent. Skin test reactions of 2–4 mm were defined as indeterminate. Before testing vaccinated volunteers, five non-immunized, healthy control volunteers with non-responder allotypes underwent ID-STs to confirm that the skin test reagents were safe and did not induce false positive ST reactions. Additional skin tests were applied in vaccinees to resolve occasional equivocal test results. In cohort A and in the five non-immunized control volunteers, the ID-ST injections of 0.02 ml were 1/1000, 1/100, and 1/10 dilutions of vaccine (1000 ␮g MAP + 1250 ␮g

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alum+100 ␮g QS-21) and adjuvant control (1250 ␮g alum+ 100 ␮g QS-21). Thus, each 0.02 ml ID injection contained the following quantity of vaccine depending on the dilution: (a) 1/1000 dilution: 0.02 ␮g MAP vaccine + 0.025 ␮g alum + 0.002 ␮g QS-21; (b) 1/100 dilution: 0.2 ␮g MAP vaccine + 0.25 ␮g alum + 0.02 ␮g QS-21; (c) 1/10 dilution: 2.0 ␮g MAP vaccine + 2.5 ␮g alum + 0.2 ␮g QS-21. Similarly, each 0.02 ml ID injection of adjuvant control contained the following quantity of reagent, depending on the dilution: (a) 1/1000 dilution: 0.025 ␮g alum + 0.002 ␮g QS-21, (b) 1/100 dilution: 0.25 ␮g alum + 0.02 ␮g QS-21, and (c) 1/10 dilution: 2.5 ␮g alum + 0.2 ␮g QS-21. In cohort B, the concentration used for the first ST was increased in an attempt to enhance the sensitivity of the skin test. Thus, we initially injected 0.02 ml of undiluted vaccine (20 ␮g MAP + 25 ␮g alum + 2 ␮g QS-21) and 1/10 dilution (2 ␮g MAP + 2.5 ␮g alum + 0.2 ␮g QS-21) and corresponding doses of control reagent. However, the 20 ␮g MAP and the corresponding control reagent induced persistent hyperpigmented skin nodules, and the control reagent induced positive ID-STs for DTH. As a result, for the second and all subsequent ID-STs, the mass of MAP vaccine injected was limited to 2 and 0.2 ␮g with corresponding doses of the control reagent. 2.7. Punch biopsies of DTH skin test sites Biopsies were taken of the DTH-reactive and control skin test sites from two volunteers who demonstrated induration ≥5 mm at the MAP vaccine site, and ≤1 mm induration to the adjuvant control reagent. Four millimeter punch biopsy specimens were placed in 10% formaldehyde fixative, and processed according to standard techniques. Paraffinembedded sections were stained with hematoxylin-eosin.

Immunoperoxidase staining was performed with anti-CD4 and anti-CD8 antibodies.

3. Results 3.1. IgG antibody responses to MAP antigen and P. falciparum sporozoites The serum antibody results from the volunteers in this study have been published [18]. In summary, the response to the malaria MAP vaccine was adjuvant dependent and required the inclusion of the co-adjuvant QS-21 to elicit optimal antibody responses. Three s.c. injections in cohort A of the MAP/alum/QS-21 vaccine formulation elicited high anti-MAP IgG ELISA titers (GMT 1:10,568) and anti-sporozoite IFA titers (GMT 1:5,511) in 11 of 12 volunteers who carried the HLA DRB1∗ 0401, DRB1∗ 1101 or DQB1∗ 0603 genotype. In most volunteers with HLA responder allotypes, antibody titers rose progressively after each immunization. By contrast, only 5 of 10 volunteers who carried random HLA genotypes developed detectable levels of IgG antibodies, and in these persons the GMTs were low (1:208 and 1:80) for anti-MAP and anti-sporozoite antibody, respectively. In cohort B, in which all volunteers were of responder genotypes, a similar trend was observed. Peak antibody titers were measured in six of the seven vaccinees 14 days after the second vaccination and declined thereafter. The one individual with no antibody response (volunteer no. 4, Fig. 1) carried both the DQB1∗ 0603 responder allele as well as the DRB1∗ 0701 allele. Poor antibody responses were also noted in cohort A volunteers of responder genotype who were heterozygous for DRB1∗ 0701 [18].

Fig. 1. The relationship between delayed-type hypersensitivity (DTH) response and anti-MAP IgG titers in cohort B. DTH was measured 1 day (open bars), 16 days (gray bars) and 85 days (black bars) after the second vaccination. The anti-MAP IgG titers (triangles) were measured 14 days after the second vaccination.

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Table 1 Delayed-type hypersensitivity (DTH) reactions to MAP vaccine skin test reagents in cohort A volunteers immunized with the malaria MAP vaccine Group, volunteer number

Class II genotypes

3, 17 3, 18 3, 19 3, 20 3, 21 3, 22 3, 23 3, 24 4/5, 25 4/5, 26 4/5, 27 4/5, 28 4/5, 29 4/5, 31 4/5, 32

DRB1∗ 0401 Rd R DRB1∗ 1101 DRB1∗ 0401 R DRB1∗ 0401, DRB1∗ 0603 DRB1∗ 0401, DRB1∗ 1101 DRB1∗ 1101 DRQ1∗ 0603 DRQ1∗ 0603 DRB1∗ 0401 R DRQ1∗ 0603 R

IFA antibody titera 5120 <80 80 10240 80 <80 10240 10240 20480 5120 10240 <80 <80 5120 <80f

Skin test response to MAP vaccine antigenb (0.2 ␮g MAP +0.25 ␮g alum +0.02 ␮g QS-21)c I = 2 × 2, E nege neg I = 5 × 5, E I = 0 × 0, E neg neg I = 10 × 15, I = 6 × 6, E I = 4 × 4, E I = 4 × 4, E neg neg neg neg

=4 × 5 =5 × 5 =0 × 0 E = 25 × 30 = 15 × 15 =5 × 6 =3 × 3

a

Reciprocal anti-sporozoite antibody titers measured 14 days after the third immunization. Diameter of induration (I) and erythema (E) in millimeters. Maximum response measured on day 2 except for volunteers 4/5, 26 and 4/5, 27 who were measured on day 7. c DTH skin test responses were negative to the 0.02 ␮g MAP/alum/QS-21 antigen, and to the 0.25 ␮g & 2.5 ␮g alum/QS-21 antigens. d R: random haplotype. e neg ≤ 1 mm. f Antibody titers measured 14 days after the second immunization. b

3.2. Delayed-type hypersensitivity skin tests In cohort A (Table 1), three (nos. 20, 24, 25) of eight vaccinees (38%) with high IFA anti-sporozoite antibody titers (1:5120–1:20,480) developed positive DTH (≥5 mm induration) using a 10 ␮g concentration of the MAP antigen. Three additional antibody responders (nos. 17, 26, 27) developed indeterminate DTH skin tests of 2–4 mm induration. DTH responses were negative (<1 mm) in seven of seven vaccinees in cohort A with no or low antibody titers (≤1:80), and in all five non-immunized volunteers. No individual developed a DTH response to the 0.02 ␮g vaccine or to the two adjuvant control solutions. In cohort B, six of seven vaccinees (86%) developed positive DTH (Fig. 1). A higher quantity (2 ␮g) of the MAP antigen was used than in cohort A (0.2 ␮g). These six individuals all had high IgG anti-MAP antibody titers (1:8127– 1:20, 480). Four of 7 volunteers (nos. 1, 3, 6, 7, Fig. 1) had positive DTH by day 29 (1 day after the second immunization), one volunteer (no. 2) converted by day 44 (16 days after the second immunization), and one volunteer (no. 5) converted by day 113 (85 days after the second immunization). On day 113, five of the six antibody responders still available for study were re-evaluated and all five still had positive DTH responses (Fig. 1). The one individual with a negative DTH (no. 4) also failed to develop IgG anti-MAP antibody (≤1:80). A typical DTH positive skin test response is illustrated (Fig. 2). Histopathologic examination of a skin biopsy specimen from a DTH site demonstrated a moderately intense superficial and pannicular mononuclear cell

Fig. 2. A positive DTH skin test response. Volunteer no. 1 was inoculated i.d. with 0.02 ml of the 100 ␮g MAP reagent (2.0 ␮g MAP + 2.5 ␮g alum + 0.2 ␮g QS-21) in duplicate 14 days after the second vaccination, and the DTH response was measured two days later. The cutaneous response to the 125 ␮g adjuvant control reagent (2.5 ␮g alum + 0.2 ␮g QS-21) was negative.

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Fig. 3. (A) Skin punch biopsy specimen from a positive DTH site (100 ␮g MAP) on the volar forearm of a vaccinee. A moderately intense superficial and deep perivascular infiltration of lymphocytes, histiocytes and some eosinophils are noted (hematoxylin-eosin stain, 10×). Some endothelial swelling is appreciated. Immunostain of the same site demonstrated CD4 T lymphocytes as the predominant component of the inflammatory infiltrate (not shown). (B) Skin punch biopsy from a control site (125 ␮g adjuvant alone). A scant superficial and mid-dermal perivascular lympho-histiocytic infiltrate is present (hematoxylin-eosin stain, 10×).

infiltrate with some eosinophils (Fig. 3A). In the center of the DTH specimen there is dermal necrosis with a mild neutrophilic infiltrate and sparse small vessel necrosis. Staining for CD4+ and CD8+ T cells demonstrated an abundance of CD4+ activity and negligible CD8+ staining (not shown). Examination of the specimen from a control test site (Fig. 3B) revealed a mild superficial and mid-dermal perivascular and mononuclear cell infiltrate with scattered eosinophils, similar in quality to the MAP vaccine reaction but markedly reduced in intensity.

4. Discussion In 1929 Herrmann and Lifschitz [3] tested 105 persons that had a diagnosis of chronic or active malaria with a crude P. falciparum antigen injected intradermally. Ninety (86%) individuals developed a DTH response. The test

was negative in an unstated number of healthy persons, and seven persons suffering from pulmonary TB or other infections. Cutaneous DTH responses against a variety of Plasmodial sporozoite and merozoite antigens have been induced in mice [27–29], rabbits [30], and monkeys [31]. This study is the first to report the development of DTH in humans to a well-characterized Plasmodial antigen. The DTH response to the MAP vaccine correlates well with anti-MAP IgG titers in vaccinated volunteers. Biopsies of the skin test sites obtained for histolopathologic staining and immuno-histochemical analysis for CD4+ mononuclear cell infiltration revealed changes compatible with cutaneous delayed hypersensitivity reactions [32]. These characteristic changes of DTH consist predominantly of infiltrating lymphocytes and macrophages, with occasional eosinophils and polymorphonuclear leucocytes. The histopathology is distinct from that of immune complex hypersensitivity (arthus reaction), which is characterized by

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prominent dermal vasculitis and dense polymorphonuclear leukocyte infiltration. The timing of the response is also consistent with DTH, peaking 48 h after the ID-ST. Investigations of DTH in cohort A demonstrated that three of eight volunteers (nos. 20, 24, 25) who developed high-titered IgG anti-sporozoite antibody also developed cutaneous DTH to a lower dose of MAP skin test antigen (0.2 ␮g) than used in cohort B (2.0 ␮g). It is possible that the three other volunteers in cohort A who developed high antibody titers (nos. 17, 26, 27), but who had a indeterminate DTH response measuring between 2–4 mm, would have developed positive DTH (≥5 mm) had 2.0 ␮g of MAP reagent been used. Of the two volunteers (nos. 23 and 31) who had high antibody titers and no cutaneous DTH, volunteer no. 23 was lost to follow up after day 2. None of the three individuals in this cohort with DTH to the 0.2 ␮g MAP/alum/QS-21 solution responded to the 0.02 ␮g solution. Furthermore, none of these three responded to the two alum/QS-21 control solutions of 0.25 and 0.025 ␮g, demonstrating the failure of low-dose adjuvants alone to elicit DTH responses. The five non-immunized controls were DTH negative, confirming that the MAP skin tests reagents (0.02 and 0.2 ␮g) do not induce non-specific cutaneous DTH. By contrast, high dose adjuvant control solution of 25 ␮g (but not 2.5 ␮g) induced positive skin tests. Although volunteers nos. 23 and 31 and the five non-immunized controls did not have screening skin tests for anergy, it is unlikely that all seven of these healthy volunteers would have been anergic to the common antigens used in such screening tests. In cohort A, DTH was found 7 months after the third inoculation of Group 3 and second inoculation of Group 4/5. Consistent with these in vivo results, peripheral blood lymphocytes and T cell lines obtained from seropositive volunteers at these time points proliferated and secreted IL-2 when stimulated with MAP or recombinant CS protein in vitro (Oliveira et al., in preparation). Volunteers nos. 21 and 28 in cohort A had the responder allele HLA DRB1∗ 0401, and volunteer no. 4 in cohort B had the responder allele HLA DRB1∗ 0603; but all three had the random allele DRB1∗ 0701, all were poor antibody responders, and all were DTH-ST negative. Low responder phenotypes associated with DRB1∗ 0701 haplotypes have also been reported for Hepatitis B vaccinees [33]. The mechanism of this allelic effect is currently unknown. No clinical grade MAP antigen free of alum was available for skin tests at the time of this study, but it appears that presence of alum and QS-21 did not interfere with the ability to elicit positive DTH skin tests. Seventy percent of the MAP antigen in the vaccine is bound to alum, and 30% of the antigen is unbound. It is unclear if the bound, unbound or both MAP antigens are active immunologically and little is known of the varieties and characteristics of malarial antigens needed for reliable DTH assays. We suspect that the MAP vaccine ID-ST reagent boosted and thus enhanced immune responses of tested volunteers, including subsequent ID-ST responses.

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The correlation of DTH skin test responses with resistance to malaria in naturally infected or experimentally immunized humans remains to be determined. DTH assays for mapping epitopes in vaccine candidate antigens have provided useful information about an HIV vaccine [7]. Such an application could contribute to malaria vaccine development by facilitating analysis of malaria-specific cellular responses in naturally infected individuals, particularly in areas of the world where access to in vitro facilities is limited. Of particular importance in this regard is the fact that DTH is mediated by TH1 cellular responses thus providing an indication of specific T cell subset responses in vivo. Peptide induced TH1 protective immunity, mediated by ␥-interferon inhibition of liver stage parasites, has been demonstrated in the rodent malaria model [14,34]. Moreover, recent Phase I/II trials of a protective recombinant CS protein vaccine detected high levels of TH1 cellular responses in the vaccinees [35,36]. The assessment of DTH in field trials will have to take into account the DTH suppressive effects of malaria infection which have been demonstrated in a variety of settings [37,38] but generally in children and adults from hypoendemic areas. Although the exact mechanism of the immunosuppressive effects of malaria are not known, recent evidence suggests that the parasite induces immune dysregulation by inhibiting the maturation of dendritic cells [39]. HIV and other infections may also interfere with DTH [40–42], as may carcinoma [43], nutritional deficiency, and metabolic abnormalities [44]. We have demonstrated that the presence of T cell functional activity reflected by a positive DTH skin test response to the MAP antigen serves as another marker for vaccine immunogenicity. Studies of DTH responses to malaria vaccine candidate antigens may provide useful information as an in vivo indication of the presence of peptide specific TH1 cells, which are potential sources of inhibitory ␥-IFN as well as helper factors for antibody responses.

Acknowledgements We gratefully acknowledge the skilled clinical assistance of Helen Secrest, R.N. and JoAnna Becker, R.N. We thank Drs. Oscar Kashala, Charlotte Kensil, and Robert Kammer, Aquila Biopharmaceuticals, Inc. for the gift of clinical lots of QS-21 and for unpublished clinical and laboratory results using QS-21 in vaccine trials. We thank Dr. C.T. Shin, Reheis, Inc. for the clinical lot of aluminum hydroxide used for skin tests. The (T1B)4 MAP vaccine was bottled at WRAIR under the supervision of Dr. Kenneth Eckels, who also helped us develop the skin test reagents. Additional thanks to Richard Pfau, M.D. for dermatopathology assistance, B. Fenton Hall for helpful discussions during the course of the study and critical review of the manuscript, and Malcolm E. Molyneux for helpful discussions on the topic. Finally, we thank the volunteers for their loyal participation.

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