Currently licensed pneumococcal conjugate vaccines (PCVs) were designed to target the 10–13 capsular polysaccharide serotypes that are the most prevalent cause of invasive pneumococcal disease (IPD), but no licensed vaccine exists that protects against all pneumococcal serotypes. Following the introduction of PCVs, pneumococcal-associated disease in young children has been significantly reduced.1 A number of studies, however, have documented replacement carriage in the nasopharynx with serotypes not included in the vaccines.2 Recently, an increase in pneumococcal disease caused by these nonvaccine serotypes has been reported.3,4 Furthermore, currently licensed PCVs are relatively expensive to produce and require substantial donor financial assistance for use in low-resource countries. Additional pneumococcal vaccines are needed that are more affordable to manufacture, provide sufficient global supply and can offer broader protection to prevent pneumococcal pneumonia and IPD, including reducing the chance for non-PCV serotype disease emergence.
Vaccines that contain proteins common to essentially all pneumococcal serotypes could potentially meet this need. A number of Phase 1 and 2 studies have been conducted or are underway to assess specific Streptococcus pneumoniae (SPn) proteins that might be included in new candidate vaccines.5–9 An alternative strategy is to use whole pneumococcal cells that contain numerous proteins and also have inherent adjuvant properties. Given the manufacturing process advantages of a whole cell vaccine (eg, high yields, low costs, etc.), if such a vaccine can be shown to induce a strong immune response, it would have the potential to provide broad individual and population protection at an affordable price. Herein, we describe a first-in-human Phase 1 clinical study to evaluate the safety, tolerability and immunogenicity of an experimental SPn whole cell vaccine candidate adsorbed to aluminum hydroxide (Alum) adjuvant (wSp) administered intramuscularly in healthy US adults.
MATERIALS AND METHODS
Study Design and Participants
This was a Phase 1 study conducted between February 13, 2012 and May 22, 2013.10 The study was reviewed and approved by the Western Institutional Review Board and conducted in compliance with the study protocol, international standards of Good Clinical Practice and the Declaration of Helsinki. The study was conducted at a single center, Comprehensive Clinical Development Northwest, in Tacoma, Washington, United States.
Participants considered for eligibility were healthy adults 18–40 years of age at the time of consent, without evidence of the following: chronic health issues; abnormal screening clinical labs; history of IPD or pneumococcal vaccination; contraindications to vaccination; recent vaccination or receipt of blood products. Forty-two participants were enrolled into 1 of 3 dose cohorts to receive 0.1, 0.3, or 0.6 mg (protein content) of wSp, or placebo (saline) using a computerized randomization block design with sequential subject assignment by data management. Pharmacy staff were unblinded and responsible for preparing and administering vaccinations. Study participants and all others involved in conducting the trial, including laboratories, remained blinded to treatment assignment.
Each dosing cohort of 14 participants received a series of 3 vaccinations at 28-day intervals with a dose escalation between cohorts. In each cohort, participants were randomized to either wSp (n = 10) or placebo (n = 4). Participants were monitored for 1 h postvaccination before release from the clinic and then self-reported local and systemic reactogenicity events (REs) using a standard diary scoring card until a follow-up visit at 7 days postvaccination. Local REs included injection site pain, tenderness, erythema, induration and itching. Systemic REs included headache, muscle pain, temperature (oral), nausea, vomiting, fatigue, diarrhea, joint aches and chills. Safety laboratory testing, schedule and symptom-directed physical examinations occurred at baseline and 7 days following each vaccination. Adverse events (AEs) were assessed at each visit and categorized by Medical Laboratory for Regulatory Activities System Organ Class (MedDRA SOC) and MedDRA Preferred Term (PT) and analyzed by study cohort, severity, duration and relationship to vaccine. An internal safety team reviewed blinded reactogenicity and laboratory results weekly. An unblinded independent data safety monitoring committee gave authority to allow dose escalation or to alter the study should a safety signal emerge. Prespecified pause rules included any serious AE (SAE), a grade 3 clinical or laboratory abnormality or >2 subjects having the same grade 3 injection site reaction or grade 2 laboratory abnormality.
SPn whole-cell antigen bulk, Lot No. 1676, was manufactured by Walter Reed Army Institute of Research from strain RM200 RX1E PdT ΔlytA (genetically modified to remove the lytA gene). The virulence factor pneumolysin gene was replaced with a gene encoding for a pneumolysoid containing 3 point mutations that abolish cytolytic activity and complement activation.11 Beta-propriolactone was utilized to inactivate cells during processing, and the final drug product [S. pneumoniae whole cell antigen (SPWCA)] was stored at <–60°C until the day of use. Dosage was specified by protein content as determined by the Kjeldahl assay, which represents approximately half the dry weight. Alum, Lot No. 1008198, was formulated at Instituto Butantan by diluting commercial Alhydrogel with normal saline and stored at 2–8°C. wSp doses were formulated on the day of vaccination by dilution in normal saline and adsorption to Alum at room temperature for 1 h before vaccination. The final formulation contained 0.6 mg of elemental aluminum per dose. Normal saline was used as the placebo. All vaccinations were given intramuscularly in the lateral deltoid muscle.
Study Hypothesis and Objectives
The primary hypothesis was that wSp would be safe and well tolerated. This objective was evaluated by solicited reactogenicity through 7 days and by unsolicited AEs through 84 days postvaccination. A secondary hypothesis was that an increase in antibody responses over baseline to wSp vaccination would be measurable. The SPWCA enzyme-linked immunosorbent assay (ELISA) was used to assess the secondary hypothesis, although it was recognized that this assay may not provide sufficient sensitivity or specificity in adult populations with significant exposure to S. pneumoniae and therefore an extensive number of other assays were included in a sequential and adaptive approach to analyze immune responses.
Assays were either developed specifically or adapted for use in this vaccine development program. Briefly, the SPWCA, Pneumolysoid (L460D), and pneumococcal surface protein A (PspA) ELISAs were developed and validated by Charles River Laboratories, Montreal, and specific antibody responses were measured following wSp vaccination. The antibodies in lymphocyte supernatant (ALS) assay measured the acute response of B-cells recently stimulated by wSp vaccination by culturing peripheral blood mononuclear cells (PBMCs) and measuring antibody responses in culture supernatants by ELISA.12 The Boston Children’s Hospital (BCH) ELISA measured antibodies to SPWCA, 8 SPn-specific proteins and pneumococcal cell wall polysaccharide. Three assays were utilized for assessing cytokine responses. An intracellular cytokine staining (ICS) assay identified the T-cell phenotype (CD4+ or CD8+) and the cytokines/cell surface markers produced following in vitro stimulation of PBMCs with SPWCA13; the Multiplex Bead Array (MBA) used a Luminex platform to measure multiple cytokines after in vitro stimulation of PBMCs with SPWCA; and interleukin 22 (IL-22) was measured by standard ELISA.
In addition, 4 functional assays were assessed for future utility in the wSp vaccine development program. Serum antibodies were assessed for their ability to neutralize wildtype pneumolysin-induced lysis of rabbit red blood cells (Ply-nAb). The validated multiplex opsonophagocytic assay (MOPA) was performed according to the methods of Romero-Steiner and assessed the ability of antibodies to facilitate the killing of 14 S. pneumoniae serotypes (6C and those contained in Prevnar13) by phagocytes.14 The surface killing assay measured opsonized pneumococci after overnight growth on blood agar plates overlaid with polymorphonuclear cells.15 An intravenous challenge model for pneumococcal sepsis, which has been described previously, was utilized as the passive protection assay (PPA).5 Briefly, mice were injected intraperitoneally with 100 μL of various dilutions of pre- and post-immunization serum. After 4 h, mice were challenged intravenously with a lethal dose of virulent serotype 3 SPn (A66.1). Mice were monitored for 14 days at 4-h intervals and scored for moribund status.
Safety data were descriptive in nature and summarized by treatment group, vaccination period and, in the case of AEs, by MedDRA SOC and PT. The intention-to-treat population was analyzed for all safety evaluations. Immunogenicity testing was by treatment group (pre- and post-baseline or change from baseline) and tested using the t test and Fisher’s exact test or other test as indicated in the results section. Analyses did not include any unmatched (pre/post vaccination) sample pairs. The study was designed to provide preliminary safety and immunogenicity data to support testing the study product in additional larger cohorts of adults and in age-descending studies, and was not statistically powered for pre-specified endpoints.
A total of 147 volunteers gave informed consent and were evaluated. Eighty-eight (88) failed to meet eligibility requirements, 17 withdrew consent and 42 were randomized into the trial. The demographics of trial participants can be found in Table 1. Compliance with the vaccination schedule was high, with only 3 participants having delays in vaccination (all at their third vaccination).
Individual local REs were reported among up to 100% of participants given varying doses of wSp and 33% of those given the placebo (Table 2). The higher rate of local REs is a common occurrence among participants receiving Alum-adjuvanted vaccines when compared with injection with saline. Maximum local reactogenicity tended to occur with the first dose of wSp, and was typically reduced with subsequent vaccinations. Nearly all the local REs were graded as mild or moderate, with duration ranging from 1 to 4 days. The most common solicited REs were pain and/or tenderness at the site of injection. Two participants who received 0.3 mg of wSp reported severe pain with the first vaccination but did not seek medical attention, and on repeat vaccinations pain was classified as mild. No events of local necrosis or abscess formation were observed. No volunteer refused further vaccination due to REs.
Solicited systemic REs were mild in nature and did not increase with repeated injections of the vaccine. No participant reported a severe systemic RE. Overall, systemic REs were less frequent than local REs with up to 50% of participants given varying doses of wSp and 25% among those given the placebo experiencing an individual systemic RE (Table 2). No obvious trends were observed across successive injections or reactogenicity type, and no one event appeared dominant when considering dosage or vaccination sequence. There were no safety laboratory changes of clinical significance observed, and fluctuations were consistent with normal day-to-day variations. Forty-five (45) unsolicited AEs were reported by 24 participants in the study (18 of 30 participants receiving wSP and 6 of 12 participant receiving placebo, respectively). Of these, 5 participants had mild AEs rated as possibly related to receipt of clinical trial material. These cases included 3 cases of injection pain, one headache that extended beyond the 7-day postvaccination period (all resolved by day 10), and one episode of dysfunctional uterine bleeding 3 days postvaccination (n = 2, n = 3 for wSp 0.3 mg and 0.6 mg, respectively). The other 40 AEs were distributed relatively equally between all 4 treatment groups, and all resolved by day 84 of the study. A single SAE (ruptured ectopic pregnancy with inadequate contraceptive method) occurred during the trial, resolved without sequelae and was deemed not related to vaccination. At the 6-month follow-up phone call, there were no AEs reported related to vaccination and no new SAEs.
SPWCA ELISA and ALS assays
The anti-SPWCA serum immunoglobulin G (IgG) response, as measured by ELISA, was chosen as the secondary endpoint since it measured responses to a broad array of antigens present in the vaccine. Anti-SPWCA IgG was assessed for each subject comparing day 0 baseline to day 28 after each vaccination (days 28, 56 and 84). No statistically significant change from baseline was detected at any wSp dose, or at any postvaccination time point using the SPWCA ELISA (Fig. 1A).
Recognizing the potential limitations of the SPWCA ELISA with respect to pre-existing antibody responses in adult subjects, the ALS assay was selected as a potential way to reduce background responses and, therefore, increase sensitivity. The ALS assay is designed to “capture” B-cells recently stimulated (eg, by vaccination) and to, therefore, measure wSp-specific stimulated antibody responses without being confounded by high pre-existing pneumococcal antibody titers.12 The ALS assay demonstrated that PBMCs from individuals vaccinated with wSp secreted statistically significantly greater concentrations of pneumococcal antibodies compared with baseline, whereas the placebo subjects showed no response (Fig. 1B).
Antibody Responses to Specific SPn Antigens Following wSp Vaccination
A variety of individual SPn antigens contained within the whole-cell vaccine may be immunogenic. We selected cohort 3 (0.6 mg and placebo) subset samples (days 0 and 84) to test prospectively using the BCH ELISAs. Median fold-rises in antibody levels were statistically significant (P < 0.05) in vaccinated participants for antibodies against 8 of the 10 antigens tested using the 2-fold cut off criteria (data not shown). Collectively, all 8 of the evaluable 0.6-mg-vaccinated subjects made a response to at least one of the pneumococcal-specific proteins (Table 3).
After analysis of the BCH ELISA results, PspA and Pneumolysoid (L460D) ELISAs were developed and validated after data lock and used to further assess immune responses among wSp vaccinees. Geometric mean titers were significantly increased at 28 days following final vaccination (day 84) with 0.6 mg wSp for Ply (2.6-fold) and PspA (2.4-fold) (P < 0.05 and P < 0.001, respectively), with Ply demonstrating a dose-dependent response (Fig. 2). No changes were observed in the placebo treatment group.
T-cell Cytokine Responses to wSp Vaccination (day 0 versus day 84)
Statistically significant increases in PBMC CD4+ ICS responses were seen for 0.6 mg wSp recipients (but not other treatment groups) with specific increases in IL-17A (P < 0.01), CD40L (P < 0.05), IL-2 (P < 0.01) and TNF-α (P < 0.05) (data not shown). For the MBA assay of PBMC culture supernatants, only IL-17A demonstrated a consistent increased response to SPWCA stimulation in vitro when comparing day 84 to baseline, with a significant increase seen following vaccination in participants receiving 0.6 mg of wSp (P < 0.01) and a nonsignificant increase in the 0.3 mg vaccinated group (Fig. 3). The IL-22 ELISA did not demonstrate a measurable increase with any treatment group (data not shown).
Functional Immune Responses to wSp Vaccination
PPA was utilized to assess functional responses for placebo- and 0.6-mg wSp-vaccinated recipients (n = 3 and n = 9, respectively) who had paired serum samples from day 0 and day 84. Testing was initially performed to compare pre- versus post-immunization responses using a 1:50 dilution of sera with additional assessment performed at dilutions of 1:10 or 1:100 for recipients noted to have a weak post-response or a strong baseline response, respectively.
A significant increase in median time to moribund state in SPn-challenged mice was seen with the sera from 6 of the 9 evaluable participants vaccinated with 0.6 mg wSp, whereas serum from 0 of 3 placebo-treated individuals provided significantly increased protection at day 84 compared with prevaccination (Table 4). One 0.6-mg-vaccinated participant had high levels of protective antibody at baseline and a change in response postvaccination could not be ascertained.
The MOPA and Ply-nAb assays were performed on the cohort-3 subset using baseline and 84 day postvaccination sera. wSp did not induce opsonophagocytic activity to any of the serotypes tested (data not shown). Four of 9 participants receiving 0.6 mg wSp demonstrated at least a 4-fold rise in Ply-nAb titer versus none of the placebo-treated participants (data not shown).
Developed through a partnership between PATH, Instituto Butantan and BCH, wSp has been shown in preclinical studies to mediate its protective responses via both T-cell (IL-17A) and B-cell immune pathways, thereby having the potential to reduce both pneumococcal carriage and disease. Pneumococcal vaccines that incorporate common protein antigens also have the potential to overcome some of the major limitations of PCVs by providing broad coverage against all serotypes while requiring less manufacturing complexity and cost. The nonencapsulated whole-cell vaccine candidate, wSp, was shown to be well tolerated based on local and systemic reactogenicity profiles in this Phase 1 study in healthy adult participants, inducing both T- and B-cell immune responses. Multiple vaccinations did not result in escalating reactogenicity, which can sometimes be seen with other whole cell vaccines such as whole cell pertussis.16
Both SPn-specific immunologic activity as well as functional (protection) activity was demonstrated most consistently at the highest dose of wSp tested (0.6 mg). Specific pneumococcal proteins known to be involved in the pathogenicity of SPn were shown to have wSp antibody-mediated immune responses. No one specific antibody response to a single antigen was identified in all recipients, although both PspA and Ply antibody responses were detected in 75% of the 0.6-mg vaccinated participants. A platform of SPn-specific assays may be needed to fully characterize the response to a whole-cell pneumococcal vaccine.17,18 In addition, similar to preclinical findings, wSp vaccination stimulated in vitro IL-17 responses from PBMC, with the 0.6-mg dose providing the best response. Another compelling feature of this Phase 1 trial was the demonstration of functional protective antibodies using both a PPA model and a pneumolysin toxin neutralization assay (Ply-nAb). Since 0.6 mg of wSp was the only dosage that induced consistent measureable immune responses over baseline, an optimal dose in adults was not likely identified in this study. Further dose escalation is warranted to achieve a response in all participants (or at least in those with low baseline antibody levels).
Trial limitations included small sample size, limited validated assays and no established immune markers for an SPn whole cell vaccine. At the time of this writing, the clinical development program of this vaccine has advanced to a Phase 2 trial in toddlers to assess dose-escalation to 1 mg (adults) and age de-escalation in toddlers with and without co-administration of Expanded Program on Immunization vaccine boosters in Kenya. Given the potential for this vaccine to impact SPn carriage (via IL-17A responses), an exploratory carriage study in these same toddlers is being conducted. Further age de-escalation to infants is planned with a goal to develop a cost-effective vaccine capable of protecting against SPn carriage, pneumonia and IPD.
Authors thank staff at Comprehensive Clinical Development Center, Tacoma Washington, United States for trial conduct; EMMES Corporation, Washington, District of Columbia, United States, for monitoring and data analysis; Susan Vintilla-Friedman for manuscript preparation; Devin Groman for editorial support; and the study participants for their contribution to this vaccine development program.
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