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. Author manuscript; available in PMC: 2013 Dec 14.
Published in final edited form as: Vaccine. 2012 Oct 22;30(52):7594–7600. doi: 10.1016/j.vaccine.2012.10.038

NDV-3, a Recombinant Alum-Adjuvanted Vaccine for Candida and Staphylococcus aureus is Safe and Immunogenic in Healthy Adults

Clint S Schmidt a, C Jo White b, Ashraf S Ibrahim c,e,f, Scott G Filler c,e,f, Yue Fu c,e,f, Michael R Yeaman c,d,e,f, John E Edwards Jr c,e,f, John P Hennessey Jr a
PMCID: PMC3513491  NIHMSID: NIHMS416272  PMID: 23099329

Abstract

The investigational vaccine, NDV-3, contains the N-terminal portion of the Candida albicans agglutinin-like sequence 3 protein (Als3p) formulated with an aluminum hydroxide adjuvant in phosphate-buffered saline. Preclinical studies demonstrated that the Als3p vaccine antigen protects mice from oropharyngeal, vaginal and intravenous challenge with C. albicans and other selected species of Candida as well as both intravenous challenge and skin and soft tissue infection with Staphylococcus aureus. The objectives of this first-in-human Phase I clinical trial were to evaluate the safety, tolerability and immunogenicity of NDV-3 at two different antigen levels compared to a saline placebo. Forty healthy, adult subjects were randomized to receive one dose of NDV-3 containing either 30 or 300 μg of Als3p, or placebo. NDV-3 at both dose levels was safe and generally well-tolerated. Anti-Als3p total IgG and IgA1 levels for both doses reached peak levels by day 14 post vaccination, with 100% seroconversion of all vaccinated subjects. On average, NDV-3 stimulated peripheral blood mononuclear cell (PBMC) production of both IFN-γ and IL-17A, which peaked at day 7 for subjects receiving the 300 μg dose and at day 28 for those receiving the 30 μg dose. Six months after receiving the first dose of NDV-3, nineteen subjects received a second dose of NDV-3 identical to their first dose to evaluate memory B- and T-cell immune responses. The second dose resulted in a significant boost of IgG and IgA1 titers in >70% of subjects, with the biggest impact in those receiving the 30 μg dose. A memory T-cell response was also noted for IFN-γ in almost all subjects and for IL-17A in the majority of subjects. These data support the continued investigation of NDV-3 as a vaccine candidate against Candida and S. aureus infections.

Keywords: vaccine, phase 1, first-in-human, Als3p, Candida, Staphylococcus aureus

Introduction

Candida sp. are a major cause of hospital acquired infections in the U.S. and worldwide [1, 2]. Candida sp. are now equivalent to enterococci as the third most frequent hospital acquired bloodstream isolates [3, 4], accounting for 10% of bloodstream infections and 11% of catheter-related infections [5]. Population-based surveys in the U.S. have reported the annual incidence of Candida bloodstream infections is approximately 20 cases per 100,000 people (60,000 cases per year) [6, 7]. In high risk/hospitalized patients, this incidence increases 50-fold [1, 6, 8, 9]. These rates represent 15 to 20-fold increases compared to two decades earlier [10–12]. In addition to hematogenously disseminated candidiasis, mucosal candidal infections are common and can be persistent in some patients, causing recurrent disease several times per year. Most notable in this respect is recurrent vulvovaginal candidiasis, which impacts 5–8% of women in the US [13].

Staphylococcus aureus is the most common cause of skin and skin structure infections [14] and endocarditis [5], and the second most common cause of bacteremia [3, 4]. S. aureus is a primary cause of a variety of hospital acquired infections, including ventilator-associated pneumonia, intravenous-catheter associated infections, post-surgical wound infections and is also a predominant cause of battlefield wound infections [15, 16]. This organism frequently causes invasive infections in neutropenic patients and those undergoing solid-organ or hematopoietic stem cell transplants [17]. Invasive infections caused by S. aureus continue to increase in frequency [18, 19]. The increase in incidence of serious infections caused by S. aureus is concerning given the high mortality associated with S. aureus bacteremia and endocarditis (15 to 40%), even with appropriate antimicrobial therapy [18, 20, 21]. In addition, over the past decade S. aureus has become increasingly resistant to available antimicrobials [22]. To date, there are no licensed prophylactic or therapeutic vaccines either for S. aureus or Candida.

The agglutinin-like sequence 3 protein (Als3p) of C. albicans is both an adhesin [23] and an invasin [24] for Candida. It also has both sequence and structural homology with cell surface proteins on Staphylococcus aureus [23]. These findings led to its evaluation as a vaccine antigen where it was demonstrated to have protective efficacy in preclinical animal models of oral, vaginal and disseminated candidiasis as well as disseminated staphylococcemia [25–27] and S. aureus skin and soft tissue infection (unpublished data). Additionally, this protective immunity was effective against several species of Candida [25] and against several clinical isolates of S. aureus [27]. Finally, the vaccine was shown to be highly immunogenic in animal models, inducing robust anti-Als3p B-cell response in mice [28], rabbits and non-human primates (unpublished data) along with robust T-cell responses in mice [29].

Based on these preclinical observations, purified Als3p bulk was manufactured under current Good Manufacturing Practices (cGMP), incorporated into formulations containing aluminum hydroxide as an adjuvant (designated as NDV-3 vaccine) and evaluated in this clinical study. The results of evaluating the safety, tolerability and immunogenicity of NDV-3 vaccine in healthy volunteers are presented below.

Methods

Study design

The study was a double-blind, placebo-controlled, ascending dose escalation study (30 and 300 μg) that enrolled healthy adults at a single study site. Vaccinations occurred on study day 0, with follow up evaluations on study days 3, 7, 14, 28, 90 and 180. A subset of vaccinees was re-consented to receive a second dose of vaccine on study day 180, with follow up visits 7, 14, and 90 days after the second dose. The lower participation rate in receiving the second dose (9 of 15 for the 30 μg dose and 10 of 15 for the 300 μg dose) was documented as primarily due to the timing of the second dose and follow-up conflicting with mid-summer personal schedules. Details of the study design and execution are provided in Supplemental Materials.

Vaccine and adjuvants

The active component of the NDV-3 vaccine is a recombinant version of the N-terminal region (416 amino acids) of the C. albicans Als3p with the addition of a six-His tag and linker sequences [26]. Als3p was produced by batch fermentation of a Saccharomyces cerevisiae expression cell line at 100 L scale, harvested by centrifugation and purified using two chromatography columns (nickel-affinity and hydrophobic interaction resins) followed by concentration, diafiltration into phosphate-buffered saline (PBS), pH 7, and filtration. The purified Als3p bulk drug substance was intact, monomeric and 99% pure by SDS-PAGE with Coomassie staining and was formulated with aluminum hydroxide at 1.0 mg Al/mL in PBS, pH 7. Two final container vaccine clinical lots were used for this study; lot 0939 (60 μg Als3p/mL) and lot 0940 (600 μg Als3p/mL). Clinical lots were stored at 2–8°C post manufacture and monitored for stability. Manufacture of the bulk drug substance and final container lots using cGMPs was conducted by Althea Technologies (San Diego, CA).

Immunogenicity analysis

Blood samples were obtained from subjects on the specified days post vaccination. Plasma and PBMCs were isolated using standardized procedures. Plasma samples were evaluated for anti-Als3 total IgG and for anti-Als3 IgA1 by standardized ELISA methodology. Results are expressed in units of dilution−1. PBMCs were evaluated by ELISpot analysis to determine the portion of cells that could be stimulated to produce IFN-γ or IL-17A (two separate assays). Results are expressed in units of spot forming units (SFU) per 106 cells. Details of the above procedures are provided in Supplemental Materials.

Statistical Analyses

Statistical analysis of assay results used non-parametric analysis using the Wilcoxon rank-sum test [30]. Evaluation of trends across groups used the Kruskal-Wallis test [31].

Results

An overview of the clinical study design (Figure S1) and execution (Table S1) are provided in Supplemental Materials.

Safety

In this study population, NDV-3 was safe and generally well-tolerated after one or two doses. Local injection site reactions to placebo (post dose 1) and vaccine and (post dose 1 and 2) are summarized in Table 1. The most common complaint was injection site pain, typically mild, lasting 1–2 days after vaccination and resolving within 2–3 days without sequelae.

Table 1.

Systemic and injection side AEs reported Days 0–7 post-vaccination regardless of causality. AEs included below reflect any AE that was reported by at least 2 vaccinees during that time frame, i.e. out of 40 total post-dose 1 and out of 19 post-dose 2.

MedDRA Preferred Term Placebo 30 μg Dose 300 μg Dose
Dose 1 N=10 Dose 1 N=15 Dose 2 N=9* Dose 1 N=15 Dose 2 N=10*
Injection site
 Erythema 10% 20%** 11%*** 0 10%**
 Induration 10% 0 0 0 20%**
 Pain 20% 73%** 100% 73%# 100%**
 Swelling 0 7% 22%*** 7% 30%**
Systemic AEs
 Diarrhea 0 7% 11% 7% 0
 Nausea 0 13% 0 0 30%
 Fatigue 10% 7% 11% 7% 40%
 Myalgia 0 0 11% 20% 20%
 Pain in extremity 0 0 0 13% 10%**
 Headache 10%** 7% 22% 7%** 30%
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*

Subjects volunteered to continue in study to receive a 2nd dose.

**

One graded as “moderate”.

#

Three graded as “moderate”.

***

One graded as “severe”.

All AEs resolved without sequelae.

The systemic and injection site adverse events (AEs) occurring in at least two study subjects after either the first or the second dose are presented in Table 1. After dose 1 each of the systemic AEs shown in Table 1 were reflected in 2–3 of the 40 subjects. After dose 2, the most common systemic AEs were fatigue and headache (5 out of 19 (26%) subjects for each). Systemic AEs were usually mild and occasionally moderate, but all resolved without sequelae within a few days. There were no notable differences in systemic AEs between the two dose levels.

Immune Response

Plasma Anti-Als3p Total IgG and IgA1

Prior to vaccination (day 0), 36 of the 40 subjects exhibited a detectable pre-existing anti-Als3p total IgG titer ranging from 114 to 2608 dilution−1, with 4 subjects showing IgG titers below the limit of detection (LOD) of the assay (<50 dilution−1). For IgA1 titers, 36 of the 40 subjects exhibited pre-existing detectable anti-Als3p IgA1 titer ranging from 102 to 6473 dilution−1, with 4 subjects showing IgA1 titers below the LOD (<50 dilution−1). Two subjects had no detectable anti-Als3p IgG or IgA1 prior to vaccination.

The geometric mean of anti-Als3p total IgG titers (Figure 1A) and IgA1 titers (Figure 1B) rose quickly after the first dose of vaccine, showing a substantial rise by day 7 post vaccination and reaching a maximum by day 14 post vaccination. The IgG and IgA1 titers from day 7 on were significantly higher for the 300 μg dose relative to the 30 μg dose (Mann-Whitney test, p<0.05) and both were beyond the range of placebo recipient titers (Mann-Whitney test, p<0.001). Antibody titers out to 180 days post vaccination showed roughly a two-fold decline from the maximum titers.

Figure 1. Plasma Anti-Als3 Total IgG and IgA1 Titers through Day 270 post vaccination.

Figure 1

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Plasma was collected from all subjects at several time points and analyzed for anti-Als3 total IgG and IgA1 titers using indirect ELISA (see “Methods”). Geomean anti-Als3p IgG (panel A) and IgA1 (panel B) values and standard deviations of the geomean are shown, though the latter are generally within the bounds of the symbol at each time point.

Following the second dose of vaccine, marked increases in anti-Als3p total IgG and IgA1 were noted, with the increase in the geomean IgG titer of the 30 μg recipients being greater than that of the 300 μg recipients.

The fold-rise of anti-Als3p total IgG titers (Figure 2A) and IgA1 titers (Figure 2B) above the pre-vaccination titers all peaked at 14 days post vaccination. The fold-rise for IgG from day 7 to 180 was significantly higher for the 300 μg dose relative to the 30 μg dose (Mann-Whitney test, p<0.05) and for both dose levels the mean fold-rise remained >7-fold above pre-vaccination titers through day 180 post vaccination. Based on a definition of seroconversion being a ≥4-fold rise in antibody titer relative to the pre-vaccination antibody titer, a single dose of NDV-3 resulted in 13% and 53% of subjects seroconverting in IgG by day 7 (30 and 300 μg dose, respectively) and 100% seroconverting by day 14 in both dose groups (see Table 2). Similar results were observed for IgA1 seroconversion. For the 300 μg dose group, seroconversion remained at 100% for IgG through day 180, although for IgA1 there was a decline from 100% after day 28. For the 30 μg dose group, the seroconversion rate for IgG declined from 100% after day 28, while IgA1 seroconversion rates declined from 100% after day 14 (Table 2).

Figure 2. Fold-Rise Increases in Plasma Anti-Als3p Total IgG and IgA1 through Day 270 post vaccination.

Figure 2

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The fold-rise in antibody titer is calculated for each subject as the ratio of the antibody titer at each time point post vaccination to the pre vaccination titer (day 0). For a given group and time point, the fold-rise is calculated as the geomean of the fold-rise values for all individuals in the group for total IgG (panel A) and IgA1 (panel B). Geomean values and standard deviations of the geomean are shown for each group and time point. The dashed line represents a 4-fold rise in antibody titer.

Table 2.

Fold rise of anti-Als3 antibody titer relative to pre-vaccination (Day 0) titers.

Dose (μg) Ig type % of subjects with ≥4-fold rise in anti-Als3 antibody titer
Day 7 Day 14 Day 28 Day 90 Day 180
30 IgG 13 100 100 93 73
300 IgG 53 100 100 100 100
30 IgA1 7 100 93 87 87
300 IgA1 60 100 100 87 80
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When study subjects were given a second dose of vaccine identical to their first, the increase in antibody titer after 14 days was relatively modest for those receiving the 300 μg dose, with increases in the GMT for IgG and IgA1 of 1.8- and 2.0-fold (data not shown). For those receiving the 30 μg dose the GMT of IgG titers after 14 days increased 4.1-fold and the GMT for IgA1 increased 2.4-fold. At either dose level, the kinetics of the decrease in IgG and IgA1 titers appears to resume about the same rate as seen after the first dose (Figure 1).

Anti-Als3 IFN-γ and IL-17A Production by stimulated PBMCs

Figure 3 provides an overview of the IFN-γ (panel A) and IL-17A (panel B) ELISpot results for subjects receiving the 30 μg dose of vaccine. Subjects at each time point were segregated into “non-responders” and “responders”, depending on whether or not their result exceeded the empirical cutoff of 25 SFU per 106 cells. For both IFN-γ and IL-17A, the maximum response was seen on day 28 post vaccination. The mean responses (SFU per 106 cells) for responders in each assay were similar on days 7 to 180, with a significantly greater number of cells producing IFN-γ than IL-17A.

Figure 3. Als3-stimulated IFN-γ and IL-17A production by PBMCs for all subjects receiving the 30 μg dose through Day 270 post vaccination.

Figure 3

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PBMCs were collected from all subjects at several time points and were analyzed for Als3-specific stimulation of IFN-γ (panel A) or IL-17A (panel B) production using ELISpot assay (see “Methods”). Values less than 2 are plotted as a cloud at the bottom of the stack so that all subjects are represented on the figure. The definition of a responder in either assay is an ELISpot result that is a ≥ 25 SFU per 106 cells difference in Als3 peptide-stimulated cultures minus unstimulated cultures (dashed line). The median value ( Inline graphic) for each data set is shown.

Figure 4 provides an overview of the ELISpot results for subjects receiving the 300 μg dose of vaccine. In this case subjects receiving 300 μg of Als3p responded more rapidly than those receiving 30 μg Als3p, i.e. by day 7, and with a greater number of cells producing both cytokines. However, the drop off of response appeared to be more rapid, with a diminished response at day 90 post vaccination for both cytokines. Again, the IFN-γ producing cells far outnumbered the IL-17A cells in the responders at all time points.

Figure 4. Als3-stimulated IFN-γ and IL-17A production by PBMCs for all subjects receiving the 300 μg dose through Day 270 post vaccination.

Figure 4

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PBMCs were collected from all subjects at several time points and were analyzed for Als3-specific stimulation of IFN-γ (panel A) or IL-17A (panel B) production using ELISpot assay (see “Methods”). Values less than 2 are plotted as a cloud at the bottom of the stack so that all subjects are represented on the figure. The definition of a responder in either assay is an ELISpot result that is a ≥ 25 SFU per 106 cells difference in Als3 peptide-stimulated cultures minus unstimulated cultures (dashed line). The median value ( Inline graphic) for each data set is shown.

Figure 5 presents ELISpot data as the percent of positive responders for each group, including placebo recipients, at each time point. While this presentation reinforces the observations described above, it also makes clear that the IFN-γ response was more robust than the IL-17A response, with a greater percent of subjects responded at each time point and a greater difference from the placebo response. There was a more rapid rise in the percentage of both IFN-γ and IL-17A responders in the 300 μg dose group versus the 30 μg dose group on day 7 post-vaccination. However, by day 28 the 30 μg dose group showed slightly higher response rates than seen in the 300 ug dose group. Furthermore, administration of the second dose at day 180 markedly increased the percentage of responders, with the IFN-γ response elevated most by the 30 μg dose and the IL-17A response elevated most by the 300 μg dose.

Figure 5. Percentage of Subjects Responding with IFN-γ and IL-17A producing PBMCs through Day 270 post vaccination.

Figure 5

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The plotted results represent the percentage of individuals in a given group and time point that had a valid IFN-γ (panel A) or IL-17A (panel B) ELISpot response that exceeded the specified cutoff (≥25 SFU per 106 cells difference in Als3 peptide-stimulated cultures minus unstimulated cultures).

Discussion

Data from this first-in-human study suggests that the NDV-3 vaccine at both 30 and 300 μg doses is generally well-tolerated. Local site reactions were fairly typical of an intramuscular vaccine formulated with an aluminum adjuvant, with no notable difference between dose levels, but with injection site pain and swelling showing a trend of increasing after dose 2. Systemic AEs at both dose levels and after both doses were generally mild and resolved quickly without sequelae. The fact that there were no serious AEs, that the mild or moderate AEs at either dose level and after both doses resolved within a few days and that there were no notable differences between the two dose levels after each dose, suggests that this protein antigen is safe in this study population (healthy adults 19–47 years old).

The immune response assays employed in this study were based on the accumulated preclinical evaluation of NDV-3 in mice that suggested that the primary B-cell responses of interest were production of total IgG and IgG2a [28] and the primary T-cell responses of interest were Als3-stimulated production of IFN-γ and IL-17A [29]. While additional Als3-stimulated immune responses could have been measured, evaluation of other responses (e.g. other antibody isotypes and Th2 responses) in mice [28] and in unvaccinated humans [32] did not present compelling evidence that these were as likely to provide future surrogate markers of protection. Furthermore, in this first study of NDV-3 in humans, sampling was confined to blood draws at defined time points to permit evaluation of the magnitude and kinetics of these immune response markers without being overly invasive. Thus, the current results set the stage for future studies i.e. localized sample collection (e.g. cervicovaginal washes), additional isotype analysis and further cytokine profiling to identify potential immune biomarkers of greatest relevance to understanding the mechanisms of NDV-3 response and potential efficacy.

A total of 73 healthy adults have been evaluated for the background presence of naturally occurring anti-Als3p antibodies [40 in this study, 13 previously [32], plus 20 additional (unpublished data)]. Anti-Als3p antibodies (IgG and IgA1) were detected in 69 (95%) of these subjects. The fact that 100% of the vaccinees (n=30) in this trial responded with a rapid rise in anti-Als3p antibody titer supports the concept that natural colonization or prior infection provides a priming effect for this recombinant Als3p antigen, allowing a single dose of vaccine to elicit an anamnestic response in most people. This finding also suggests that those presenting as below the limit of detection (“<LOD”) based on testing a 50-fold dilution of serum might have shown a quantifiable response if a lower fold dilution of serum were evaluated.

As shown in Figures 1 and 2, a single dose of NDV-3 induced rapid and robust anti-Als3p IgG and IgA1 responses. Although antibody titer is considered either a correlate of protection or at least a surrogate marker of protection for many human vaccines [33], it is too early in the clinical development of NDV-3 to advance a hypothesis on the clinical significance of the antibody response. Even so, the kinetics of antibody titer rise and fall and seroconversion rates (Table 2) present a profile that is very attractive for addressing a healthcare-associated pathogen, where protection after one dose may be a critical application of a vaccine product. Preliminary data indicate that antibodies produced in subjects vaccinated with NDV-3 promoted a variety of functional responses, e.g. opsonophagocytic killing (OPK) of both C. albicans and S. aureus, blocking C. albicans adherence to, invasions of, and damage to host cells (data not shown).

Although there was a general increase in antibody titers after a second dose of NDV-3, both dose levels resulted in almost the same GMT for IgG as seen after the first dose of 300 μg (Figure 1). This response may reflect a maximum capacity for IgG response to this antigen. The option of one dose of a relatively high antigen dose level or two doses of a much lower antigen dose level could be a critical consideration not only for specific clinical indications, but also if Als3 was combined with other antigens in a combination vaccine product as the higher antigen dose level could lead to immune interference in combination antigens.

In contrast, the impact of the second dose on the GMT for IgA1 appears to be different; the post dose 2 peak titers for each dose level are similar to what was achieved after the first dose at each dose level (Figure 1). This response suggests that the ultimate dosing strategy will also depend in part on whether IgG and/or IgA are responsible for functional OPK activity.

NDV-3 also induced a substantial T-cell response, as evidenced by the post-vaccination increase in the number of PBMCs producing IFN-γ and IL-17A upon Als3-peptide stimulation. The time course of the number of cells producing cytokines (Figures 3 and 4) shows that the response peaked by day 28 post-vaccination, declined out to day 180, and then rebounded upon administration of a second dose to produce a median response that was similar to or greater than that seen after the first dose. When examined as the percentage of positive responders (Figure 5) it appears that, at least for IFN-γ, the first and second dose of NDV-3 produced a similar percentage of positive responders within weeks of dose administration. This finding suggests that memory T-cells induced by natural exposure to Candida or NDV-3 have a rapid recall response on re-exposure to Als3p. Of note, the 300 μg Als3p dose appeared to induce a more robust early response than the 30 μg dose, as there were a smaller percentage of positive responders after day 7 in the subjects who received the 30 μg Als3p dose. A similar trend was seen for the IL-17A response after the first dose, though the overall percentage of responders was lower than for IFN-γ. This outcome may suggest that the IL-17A response to the first NDV-3 dose is limited in human vaccinees. Analysis of the post dose 2 data suggests that 300 μg Als3p produces a more robust IL-17A response. Therefore, a second dose may be beneficial if an IL-17A response proves essential for protection in humans. It is interesting to note that recent work by Netea et al [34] suggests that infection by live, but not heat-killed, Candida suppresses the Th17 immune response.

The degree of T-cell stimulation by NDV-3 is remarkable for a protein antigen with an aluminum adjuvant if for no other reason than the lack of precedent for measuring human T-cell responses from a recombinant protein antigen. For example, IFN-γ producing cells reached a median level of >200 SFU/106 cells at most time points evaluated and rose to >1000 SFU/106 cells in some subjects. These levels are on the order of those seen with DNA and viral vector vaccines in humans [35, 36] and are suggestive of a robust Th1 response induced by this vaccine. In addition, a majority of subjects demonstrated an elevation of IL-17A-producing cells, suggesting NDV-3 induces a mixed Th1-Th17 immune enhancement. This same pattern was previously seen in mice, where Als3p-specific IFN-γ and IL-17-secreting T cells appear to be the protective mechanism of NDV-3 against both Candida and S. aureus [36]. Considerable recent attention has been given to IL-17 secreting CD4+ (Th17) cells and their potential role in vaccine-induced immunity to a diverse array of bacteria and viruses in preclinical models [37].

Animal models may not parallel the human condition of universal pre-exposure to commensal pathogen (Candida or S. aureus) prior to the onset of disease. Moreover, most humans appear to have a modest but pre-existing Als3-specific IFN-γ and IL-17A response that can be enhanced upon vaccination. While it is conceivable that an enhanced non-specific IL-17 response could be detrimental, (e.g. inducing organ-specific and systemic autoimmune diseases [38]), the lack of AEs observed in this study is a preliminary indication that this is not an issue with this vaccine in this healthy adult population. The precise role of an enhanced antigen-specific Th17 immune response in humans in combating candidal or staphylococcal disease is not known. However, it is clear that a genetic deficiency of Th17 immune pathway results in greater susceptibility of humans to infection by these and other pathogens [38]. These collective findings support continued exploration of the Th1 and Th17 responses to NDV-3 as a potentially important contributors to vaccine efficacy for Candida and S. aureus [39].

The decision to use a saline placebo for this study was based in part on preclinical studies with NDV-3 in non-human primates which showed no enhancement of Als3-specific responses in animals receiving alum-containing placebo (unpublished data) and a decision to not impose on placebo recipients the local site AEs that are routinely associated with administration of aluminum hydroxide. This is a typical design feature for Phase 1 non-viral vaccine studies conducted in the US over the past decade (see www.clintrials.gov). While done with good intent and acceptable to the FDA, use of a saline placebo does limit knowledge of the “control” response to all vaccine components in the absence of the specific antigens. When dealing with antigens from commensal organisms, for which study participants are expected to be pre-primed, as well as antigens and adjuvants that each induce inflammatory responses, there is the risk that use of a vaccine containing an adjuvant as compared to a saline control will mistake non-specific responses (due to the adjuvant) for antigen-specific responses. Based on the non-human primate study, this risk seemed minimal.

In a similar vein, no first-in-human study can cover all of the biologically relevant questions about the impact of a new vaccine candidate on human recipients. Questions about gut microbial impact, other coincident pro-inflammatory responses that were not monitored in this study and the magnitude and cell-type specific origin of the cytokine responses were simply beyond the scope of this first-in-man study and will be considered, along with the use of an adjuvant-containing placebo, in future studies.

Currently S. aureus vaccines appear to be a consensus target for development by large pharmaceutical companies, with Pfizer, GlaxoSmithKline, Merck, sanofi pasteur, Novartis and others showing activity in this field. The mainstream of development has focused on antibody production and opsonophagocytic function rather than on T-cell responses [40]. Results reported here provide a sound basis for further exploration of the potential importance of T-cells in the protective immune response to this bacterium, including MRSA. Future studies with the investigational NDV-3 vaccine will evaluate whether the humoral response and/or cellular and cytokine responses are more effective in prevention of staphylococcal and/or candidal infections in Phase II clinical trials.

Supplementary Material

01

Highlights for NDV-3 manuscript.

  • NDV-3 is safe and generally well-tolerated in healthy adults

  • NDV-3 elicits quick and robust B- and T-cell immune responses

  • A single dose induces an anamnestic rather than priming immune response

  • T-cell response includes increases in PBMCs producing IFN-γ and/or IL-17A

  • A second dose further enhances the B- and T-cell immune response

Acknowledgments

Funding

This work was supported in part by the National Institutes of Health to J.E.E. [R01 AI19990, R01 AI063382 & 2R42 AI071554-02A1], which provided the majority of funding to develop the production process and manufacture clinical supplies, and in part by the Department of the Army, [J.E.E, W81XWH-10-2-0035 & J.P.H, W81XWH-11-1-0686], which provided the majority of funding for development of clinical assays and conducting the clinical study. Additional acknowledgements and statements of involvement are included in Supplementary Materials.

The authors thank Erica Marchus, Mary Lizakowski and Elizabeth Zupi for their extensive efforts supporting the clinical sampling and immune response testing.

Footnotes

Clinicaltrials.gov Identifier: NCT01273922.

Conflict of Interest

C.S.S., A.S.I., S.G.F., Y.F., M.R.Y., J.E.E. and J.P.H. own equity in NovaDigm Therapeutics, Inc., which is developing vaccine technologies.

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References

  • 1.Rangel-Frausto MS, et al. National epidemiology of mycoses survey (NEMIS): variations in rates of bloodstream infections due to Candida species in seven surgical intensive care units and six neonatal intensive care units. Clin Infect Dis. 1999;29:253–258. doi: 10.1086/520194. [DOI] [PubMed] [Google Scholar]
  • 2.Diekema DJ, et al. Epidemiology of candidemia: 3-year results from the emerging infections and the epidemiology of Iowa organisms study. J Clin Microbiol. 2002;40(4):1298–302. doi: 10.1128/JCM.40.4.1298-1302.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Wisplinghoff H, et al. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004;39(3):309–17. doi: 10.1086/421946. [DOI] [PubMed] [Google Scholar]
  • 4.Wisplinghoff H, et al. Current trends in the epidemiology of nosocomial bloodstream infections in patients with hematological malignancies and solid neoplasms in hospitals in the United States. Clin Infect Dis. 2003;36(9):1103–10. doi: 10.1086/374339. [DOI] [PubMed] [Google Scholar]
  • 5.Arnow PM, Quimosing EM, Beach M. Consequences of intravascular catheter sepsis. Clin Infect Dis. 1993;16:778–784. doi: 10.1093/clind/16.6.778. [DOI] [PubMed] [Google Scholar]
  • 6.Kao AS, et al. The epidemiology of candidemia in two United States cities: results of a population-based active surveillance. Clin Infect Dis. 1999;29(5):1164–70. doi: 10.1086/313450. [DOI] [PubMed] [Google Scholar]
  • 7.Hajjeh RA, et al. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol. 2004;42(4):1519–27. doi: 10.1128/JCM.42.4.1519-1527.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Nolla-Salas J, et al. Candidemia in non-neutropenic critically ill patients: analysis of prognostic factors and assessment of systemic antifungal therapy. Study Group of Fungal Infection in the ICU. Intensive Care Med. 1997;23(1):23–30. doi: 10.1007/s001340050286. [DOI] [PubMed] [Google Scholar]
  • 9.Rennert G, et al. Epidemiology of candidemia--a nationwide survey in Israel. Infection. 2000;28(1):26–9. doi: 10.1007/s150100050006. [DOI] [PubMed] [Google Scholar]
  • 10.Fraser VJ, et al. Candidemia in a tertiary care hospital: epidemiology, risk factors, and predictors of mortality. Clin Infect Dis. 1992;15(3):414–21. doi: 10.1093/clind/15.3.414. [DOI] [PubMed] [Google Scholar]
  • 11.Karlowsky JA, et al. Candidemia in a Canadian tertiary care hospital from 1976 to 1996. Diagn Microbiol Infect Dis. 1997;29(1):5–9. doi: 10.1016/s0732-8893(97)00068-0. [DOI] [PubMed] [Google Scholar]
  • 12.Richet HM, et al. Risk factors for candidemia in patients with acute lymphocytic leukemia. Rev Infect Dis. 1991;13(2):211–5. doi: 10.1093/clinids/13.2.211. [DOI] [PubMed] [Google Scholar]
  • 13.Sobel JD, et al. Maintenance fluconazole therapy for recurrent vulvovaginal candidiasis. N Engl J Med. 2004;351(9):876–83. doi: 10.1056/NEJMoa033114. [DOI] [PubMed] [Google Scholar]
  • 14.Carratala J, et al. Factors associated with complications and mortality in adult patients hospitalized for infectious cellulitis. Eur J Clin Microbiol Infect Dis. 2003;22(3):151–7. doi: 10.1007/s10096-003-0902-x. [DOI] [PubMed] [Google Scholar]
  • 15.Calhoun J. Extremity War Injuries: State of the Art & Future Directions. Session III: Antibiotics and War Wounds. American Academy of Orthopedic Surgeons 2006 Annual Meeting; 2006; Chicago, IL. [Google Scholar]
  • 16.Aronson NE, Sanders JW, Moran KA. In harm’s way: infections in deployed American military forces. Clin Infect Dis. 2006;43(8):1045–51. doi: 10.1086/507539. [DOI] [PubMed] [Google Scholar]
  • 17.Moreillon P, Que Y-A, Glauser MP. Staphylococcus aureus (including staphylococcal toxic shock) In: Mandell GL, Bennett JE, Dolin R, editors. Principles and Practice of Infectious Diseases. Elsevier; Philadelphia, PA: 2005. pp. 2321–2351. [Google Scholar]
  • 18.Hoen B, et al. Changing profile of infective endocarditis: results of a 1-year survey in France. Jama. 2002;288(1):75–81. doi: 10.1001/jama.288.1.75. [DOI] [PubMed] [Google Scholar]
  • 19.Frimodt-Moller N, et al. Epidemiology of Staphylococcus aureus bacteremia in Denmark from 1957 to 1990. Clin Microbiol Infect. 1997;3(3):297–305. doi: 10.1111/j.1469-0691.1997.tb00617.x. [DOI] [PubMed] [Google Scholar]
  • 20.Benn M, Hagelskjaer LH, Tvede M. Infective endocarditis, 1984 through 1993: a clinical and microbiological survey. J Intern Med. 1997;242(1):15–22. doi: 10.1046/j.1365-2796.1997.00153.x. [DOI] [PubMed] [Google Scholar]
  • 21.Hogevik H, et al. Epidemiologic aspects of infective endocarditis in an urban population. A 5-year prospective study. Medicine (Baltimore) 1995;74(6):324–39. doi: 10.1097/00005792-199511000-00003. [DOI] [PubMed] [Google Scholar]
  • 22.Chambers HF, DeLeo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews Microbiology. 2009 Sep;7:629–641. doi: 10.1038/nrmicro2200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sheppard DC, et al. Functional and structural diversity in the Als protein family of Candida albicans. J Biol Chem. 2004;279:30480–9. doi: 10.1074/jbc.M401929200. [DOI] [PubMed] [Google Scholar]
  • 24.Filler SG, Sheppard DC. Fungal invasion of normally non-phagocytic host cells. PLoS Pathog. 2006;2(12):e129. doi: 10.1371/journal.ppat.0020129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ibrahim AS, et al. The anti-Candida rAls1p-N vaccine is broadly active against disseminated candidiasis. Infect Immun. 2006;74:3039–3041. doi: 10.1128/IAI.74.5.3039-3041.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Spellberg BJ, et al. Efficacy of the anti-Candida rAls3p-N or rAls1p-N vaccines against disseminated and mucosal candidiasis. J Infect Dis. 2006;194(2):256–260. doi: 10.1086/504691. [DOI] [PubMed] [Google Scholar]
  • 27.Spellberg B, et al. The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus. Infect Immun. 2008;76(10):4574–80. doi: 10.1128/IAI.00700-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lin L, et al. Immunological surrogate marker of rAls3p-N vaccine-induced protection against Staphylococcus aureus. FEMS Immunol Med Microbiol. 2009;55(3):293–5. doi: 10.1111/j.1574-695X.2008.00531.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lin L, et al. Th1-Th17 cells mediate protective adaptive immunity against Staphylococcus aureus and Candida albicans infection in mice. PLoS Pathog. 2009;5(12):e1000703. doi: 10.1371/journal.ppat.1000703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mann HB, Whitney DR. On a test whether one of two random variables is stochastically larger than the other. Annals of Mathematical Statistics. 1947;18:50–60. [Google Scholar]
  • 31.Cuzick J. A Wilcoxon-type test for trend. Statistics in Medicine. 1985;4:87–90. doi: 10.1002/sim.4780040112. [DOI] [PubMed] [Google Scholar]
  • 32.Baquir B, et al. Immunological reactivity of blood from healthy humans to the rAls3p-N vaccine protein. J Infect Dis. 2010;201(3):473–7. doi: 10.1086/649901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine Immunol. 2010;17(7):1055–65. doi: 10.1128/CVI.00131-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Cheng SC, et al. Candida albicans dampens host defense by downregulating IL-17 production. J Immunol. 2010;185(4):2450–7. doi: 10.4049/jimmunol.1000756. [DOI] [PubMed] [Google Scholar]
  • 35.Asmuth DM, et al. Comparative cell-mediated immunogenicity of DNA/DNA, DNA/adenovirus type 5 (Ad5), or Ad5/Ad5 HIV-1 clade B gag vaccine prime-boost regimens. J Infect Dis. 2010;201(1):132–41. doi: 10.1086/648591. [DOI] [PubMed] [Google Scholar]
  • 36.Nicholson O, et al. Safety and Immunogenicity of the MRKAd5 gag HIV Type 1 Vaccine in a Worldwide Phase 1 Study of Healthy Adults. AIDS Res Hum Retroviruses. 2010 doi: 10.1089/aid.2010.0151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Lin Y, Slight SR, Khader SA. Th17 cytokines and vaccine-induced immunity. Semin Immunopathol. 2010;32(1):79–90. doi: 10.1007/s00281-009-0191-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Curtis MM, Way SS. Interleukin-17 in host defence against bacterial, mycobacterial and fungal pathogens. Immunology. 2009;126(2):177–85. doi: 10.1111/j.1365-2567.2008.03017.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Kaslow DC, Shiver JW. Clostridium difficile and methicillin-resistant Staphylococcus aureus: emerging concepts in vaccine development. Annu Rev Mol. 2010;62(6):1–15. doi: 10.1146/annurev-med-051109-101544. [DOI] [PubMed] [Google Scholar]
  • 40.Proctor RA. Is there a future for a Staphylococcus aureus vaccine? Vaccine. 2012;30(19):2921–7. doi: 10.1016/j.vaccine.2011.11.006. [DOI] [PubMed] [Google Scholar]

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