Immunological surrogate marker of rAls3p-N vaccine-induced protection against Staphylococcus aureus

Dear Editor,

We have reported that a vaccine based on the recombinant N-terminus of the candidal surface adhesin, Als3p (rAls3p-N), induced cross-kingdom protection against otherwise lethal intravenous infection caused by the bacterium, Staphylococcus aureus (Spellberg et al., 2008). Vaccine efficacy was mediated by CD3 lymphocytes, predominantly with a T helper type 1 (Th1) phenotype; humoral immunity was neither necessary nor sufficient to mediate protection. Nevertheless, because Th1 cells do provide help to B cells and stimulate production of antibody, and antibody titers are elevated even during polarized type 1 immune responses (Mosmann et al., 1986; Smith et al., 2000), we hypothesized that antibody titers could serve as surrogate markers of protection for the rAls3p-N vaccine, despite the fact that the vaccine mediates protection by inducing cell-mediated immunity.

To determine the potential for antibody titers to serve as accurate surrogate markers of protection for our antiS. aureus vaccine, we studied serum banked from 32 mice vaccinated and infected in the previously published study (Spellberg et al., 2008). Antibody titers were determined by enzyme-linked immunosorbent assay, as previously described (Spellberg et al., 2005, 2006), and compared with the known time to death from infection. A single dose of vaccine plus 0.1% aluminum hydroxide adjuvant [Al(OH)₃] resulted in a >20-fold increase in median titers of both total immunoglobulin G (IgG) and IgG_2a antibodies compared with Al(OH)₃ alone (IgG titers = 1 : 4800 vs. 1 : 200; IgG_2a titers = 1 : 1600 vs. 1 : 50, P < 0.01 for both comparisons). Two doses of vaccine resulted in an additional 10-fold rise in median titers of IgG antibodies [IgG titers = 1 : 51 200 vs. 1 : 200; P < 0.01 vs. both Al(OH)₃ alone and one dose of vaccine].

Surviving vaccinated and adjuvant control mice had higher anti-rAls3p-N IgG (12 800 vs. 200; P = 0.03) and IgG_2a (3200 vs. 200; P = 0.002) titers than those that died from infection. However, IgG titers were similar between vaccinated mice (exclusive of adjuvant control mice) that did or did not survive subsequent infection (25 600 vs. 25 600; P = 0.9). Furthermore, there was no correlation between individual IgG antibody titers and time to death when comparing all mice (ρ = 0.2, P = 0.1), all vaccinated mice [including both one and two doses of rAls3p-N plus Al(OH)₃] (ρ = 0.01, P = 0.7), or in mice vaccinated with one dose (ρ = 0.2, P = 0.4) or with two doses (ρ =−0.2, P = 0.99).

In contrast, in vaccinated mice (exclusive of adjuvant control mice) anti-rAls3p-N IgG_2a titers were higher in survivors than nonsurvivors (4800 vs. 800; P = 0.01). Furthermore, IgG_2a titers correlated with time to death (ρ = 0.4, P = 0.02), even when excluding adjuvant control mice and analyzing only mice vaccinated with either one or two doses (ρ = 0.7, P = 0.003), or mice vaccinated with one dose (ρ = 0.7, P = 0.049), or with two doses (ρ = 0.8, P = 0.02) of rAls3p-N plus Al(OH)₃.

Similarly, the ratio of IgG_2a/IgG antibody titers in individual mice vaccinated with rAls3p-N plus Al(OH)₃ was higher in survivors than nonsurvivors (median ratio = 0.5 vs. 0.03; P = 0.049). IgG_2a/IgG ratios correlated with time to death when analyzing just mice vaccinated with either one or two doses (ρ = 0.5, P = 0.02), or in mice vaccinated with one dose (ρ = 0.8, P = 0.03), or with two doses (ρ = 0.8, P = 0.02) of rAls3p-N plus Al(OH)₃.

No IgG titer had a positive or negative predictive value, or overall accuracy, of >70%, at predicting survival in vaccinated mice (exclusive of adjuvant control mice) (Table 1). In contrast, an IgG_2a titer of ≥3200 had 86% positive and negative predictive values, and an overall accuracy of 86% for survival. An IgG_2a titer of ≥1 : 6400 had 100% positive predictive value for survival, but the negative predictive value fell to 64%, resulting in an overall accuracy of 73%.

Table 1.

Accuracy of antibody titer cut-offs for predicting survival in mice vaccinated with one or two doses of rAls3p-N+Al(OH)₃ and infected with Staphylococcus aureus

	Sensitivity (%)	Specificity (%)	PPV (%)	NPV	Accuracy (%)
IgG titers
≥1600	100	0	56	NA*	56
≥3200	89	0	53	0%	50
≥6400	78	29	58	50%	56
≥12 800	67	43	60	50%	56
≥25 600	67	43	60	50%	56
≥51 200	44	57	57	44%	50
≥102 400	11	71	33	38%	38
IgG2a titers
≥1600	88	57	70	80%	73
≥3200	86	86	86	86%	86
≥6400	50	100	100	64%	73
≥12 800	38	100	100	58%	67
≥25 600	25	100	100	54%	60

^*No vaccinated mice had titers <1600; hence, the NPV = 0/0.

Sensitivity, number of surviving mice with titers greater than or equal to the cut-off/number of all surviving mice; specificity, number of mice that died with titers less than the cut-off/number of all mice that died; PPV, positive predictive value, which is the percentage of mice with titers greater than or equal to the cut-off that survived; NPV, negative predictive value, which is the percentage of mice with titers less than cut-off that died; accuracy = [(number of mice with titers greater than or equal to the cut-off that survived infection)+(number of mice with titers less than the cut-off that died from infection)/(all mice)]. NA, not applicable.

In this set of experiments, humoral immune responses served as accurate surrogate markers of anti-rAls3p-N vaccine protection even though the mechanism of the vaccine has been previously established to be independent of humoral immunity. In a recent review, Plotkin (2008) emphasized the difference between a ‘correlate’ and a ‘surrogate’ of vaccine protection. He defined a correlate as ‘A specific immune response to a vaccine that is closely related to protection against infection, disease, or other defined end point,’ while he defined a surrogate as ‘A quantified specific immune response to a vaccine that is not in itself protective but that substitutes for the true (perhaps unknown) correlate.’ Our data are concordant with Plotkin’s definition of a surrogate marker of vaccine protection, and underscore that a surrogate of protection in a set of experiments does not necessarily imply a connection between the immune assay read-out and the mechanism by which the vaccine induces protection.

We analyzed IgG_2a titers as a subset of IgG titers because interferon-γ(IFN-γ) induces class switching to IgG_2a (Snapper et al., 1988; Stevens et al., 1988), and hence IgG_2a antibodies are produced in the setting of type 1 immunity (Spellberg & Edwards, 2001). Although Al(OH)₃ is not typically considered a type 1-polarizing adjuvant, we have previously reported that when administered with rAls3p-N, Al(OH)₃ adjuvant resulted in strong priming of type 1 inflammatory cytokines in reactive murine splenocytes (Lin et al., 2008). Furthermore, a recent study reported that a major mechanism by which aluminum adjuvants enhance immune responses was by direct agonism of an intracellular pattern recognition receptor, Nalp3 (Eisenbarth et al., 2008). Activation of Nalp3 by aluminum particles resulted in expression of several cytokines, including interleukin-1β (IL-1β) and IL-18, which are proinflammatory. Indeed IL-18 is a powerful inducer of IFN-γ expression, and hence can direct subsequent Th1 cytokine production (Sugawara, 2000). These results suggested that aluminum adjuvants have the potential to promote both Th1 and Th2 immune responses, and are concordant with our finding of strong induction of IgG_2a antibody titers in mice vaccinated with Al(OH)₃ adjuvant.

Our data support the concept that facile measurement of antibody titers, particularly of subsets induced by proinflammatory cytokines, may also serve as useful surrogate markers of protection even for vaccines that function by inducing cell-mediated, rather than humoral, immunity.