Immunity to invasive diseases caused by Haemophilus influenzae type b (Hib) correlates with the presence of serum antibodies reactive with the capsular polysaccharide (PS). 1, 2 These antibodies are acquired in an age-dependent manner, and their expression is presumed to result from natural exposure to either Hib or Hib PS cross-reactive organisms 3–5 and the concomitant maturation of the B cell compartment. Antibodies to Hib PS can directly mediate opsonic activity and complement-mediated bactericidal activities. These functional activities are the primary effector mechanisms of protection against developing invasive Hib disease. Evaluation and licensure of new Hib vaccines have focused on their capacity to elicit serum Hib PS-specific antibodies. Much emphasis in this field has been placed on assigning a minimum protective titer of antibody, 6–10 and discussions have centered around the issue of whether serum anti-Hib PS antibody concentrations of 0.15 or 1.0 μg/ml more accurately reflect the true benchmark of protection. Although these discussions have been heated, they have not necessarily been illuminating, and remarkably they more often than not neglect a consideration of antibody quality (avidity) 10–13 or the role of vaccine-induced 14–16 or natural priming.
Recently attention has focused on the possible role of conjugate vaccine-induced immunologic memory in conferring protection against invasive disease. 17 This attention was driven by the observations that immunization with Hib conjugate vaccines, but not with plain Hib PS vaccine, 18 primes for the ability to elicit memory-type (recall) antibody responses on subsequent exposure to Hib PS. 14, 15 In 1987 one of us raised the hypothesis that the ability to respond rapidly upon exposure to Hib bacteria might be an important second mechanism of protection, particularly in infants with low serum antibody responses to Hib conjugate vaccination, or those whose antibody concentrations had declined below the protective threshold. 15 More recently some have argued that immunologic memory and not the absolute amount of serum antibody may be a more reliable indicator of a durably protected state. 17, 19, 20 The timeliness of this topic has been further enhanced by the findings that certain combination vaccines, most notably the combination of Hib PS conjugates with acellular pertussis and diphtheria and tetanus toxoids, resulted in lower anti-Hib PS antibody responses compared with those achieved by simultaneous but separate injections of Hib PS conjugate and acellular pertussis and diphtheria and tetanus toxoids (reviewed in Reference 17). However, these combination vaccines prime for robust memory antibody responses to plain Hib PS vaccine 20, 21 or Hib PS conjugate vaccine. 22 Although the mechanism(s) of this interference in eliciting serum antibody responses is poorly understood, the data raise concern that the use of such combined Hib vaccines could compromise the induction of protective immunity to Hib. Thus a state of uncertainty exists in the Hib field as to the relative roles of memory and humoral antibody in conferring protection. This issue is important not only for understanding the fundamental mechanisms of immunity to encapsulated pathogens but also in evaluating and licensing the increasing number of combined vaccines.
Another salvo was fired into this arena with the publication of a report by Anderson et al., 23 who presented serologic findings that they interpreted as indicating that immunologic memory elicited by natural priming does not confer protection against invasive Hib disease. In this commentary we provide an alternative interpretation of their data. We suggest that the data do not provide sufficient basis to exclude memory, induced either by natural priming or by Hib conjugate vaccination, as an important mechanism contributing to protection against Hib disease.
Anderson et al. used a radioantigen binding assay to measure serum anti-Hib PS antibodies in 47 children who were hospitalized with Hib meningitis between the years of 1971 and 1973, a time before introduction of Hib vaccination. In agreement with previous observations, 24–26 they observed a direct relationship between patient age and the ability to respond to infection with the production of serum antibodies to Hib PS. Within the first month of admission only 2 of 27 patients <18 months of age (7%) produced robust anti-Hib PS antibodies in response to infection, whereas 12 of 20 patients ≥18 months of age (60%) produced high levels of serum antibodies. The major finding of this study was that many of the older patients appeared to produce a rapid rise in serum anti-Hib PS antibodies in response to infection. This result is consistent with an immunologically primed state, and confirms previous observations made by one of us in 1977. 26 From these data Anderson et al. concluded that because these older patients developed invasive disease despite their natural priming, memory may not be sufficient to confer protection. There are two underlying issues in this seemingly straightforward data set. First is the issue of whether the response patterns observed in this cohort provide sufficient evidence for anamnestic antibody responses in these patients. The second and perhaps more important issue concerns the conclusion of Anderson et al. that failure of memory in this subset of patients implies memory is not a mechanism of protection.
With respect to the first issue, Anderson’s data clearly show that the majority of the older patients were capable of producing anti-Hib PS antibodies in response to their infection and that the concentrations of these antibodies were increasing rapidly during the first week after admission to the hospital. What is less clear is whether these antibodies represented a primary or a memory antibody response to Hib infection. In none of the patients reported by Anderson et al. was the incubation period between encountering Hib bacteria (colonization) and development of invasive Hib disease known. Nor was it known how long the children were ill before they were admitted to the hospital. The incubation period and the duration of illness before being admitted to the hospital could have been quite variable. Thus it is not possible to distinguish between a primary antibody response to an infection beginning 1 to 2 weeks before hospital admission and a secondary/memory antibody response to a recently acquired infection.
Even if one accepts the proposition that many of the patients reported by Anderson et al. were naturally primed at the time of initial Hib encounter, the sweeping conclusion that immunologic memory is not protective is problematic. As described below it is important to consider two variables: the prevalence of immunologic memory in the population older than 18 months to 2 years; and the efficacy of immunologic memory in conferring protection against developing invasive disease.
Before the Hib vaccine era the age-dependent occurrence of Hib disease was well-documented. 27, 28 The high incidence of disease in infants <18 months of age was attributed to absence of protective concentrations of serum anti-Hib PS antibodies; conversely the substantial decline in incidence after age 18 months was attributed to acquisition of serum antibodies from natural exposure to Hib or cross-reacting bacteria. 1, 7, 29 Considerable experimental data 30, 31 and observations from clinical trials with passively administered immunoglobulin 32 demonstrate that serum anti-Hib PS antibodies alone can confer protection against Hib disease. However, the actual protective antibody concentration is poorly understood, and the estimate of 0.15 μg/ml as the protective concentration was largely inferred from seroepidemiologic studies demonstrating that age acquisition of such antibody titers coincided with decline in the incidence of Hib disease. 6, 7 The problem of attributing protection solely from natural acquisition of these low levels of serum anticapsular antibody is complicated by the linkage between acquisition of anti-Hib PS antibody by natural priming and the responsiveness to plain Hib PS. Although the ability to produce serum antibodies in response to Hib PS, a so-called T cell-independent antigen, is typically considered to be an indicator of intrinsic B cell maturation, this acquisition of responsiveness could reflect the generation of memory by natural priming. There is growing evidence that at least in adults, much of the anticapsular B cell population is in the memory state because the antibodies elicited by vaccination are isotype-switched and hypermutated, features indicative of a secondary antibody response. 33–36 Similar data are not yet available in children; but it is likely that in the absence of vaccination, the interplay between B cell maturation, natural priming, the elaboration of serum antibodies and the development of memory all contribute to the acquisition of immunity to Hib.
If memory plays a fundamental role in durable protection, particularly when antibody concentrations have fallen below the protective threshold, then is it reasonable to expect that memory should be 100% efficacious in preventing disease? The success or failure of memory responses to confer protection in the population would be expected to follow a frequency distribution. Just as vaccines are rarely if ever perfectly efficacious in a population, so too memory is imperfect, particularly when the infecting inoculum is high and the incubation period is short. As seen in Table 1, if immunologic memory to Hib PS were present in 75% of the population (a reasonable value for children ≥18 months of age based on the ability of this age group to respond to plain Hib PS vaccination) and if memory alone were 90% efficacious in preventing invasive Hib disease, then 69% of patients (cases) with disease would be expected to show evidence of memory. If efficacy of memory in preventing disease were 90% and memory were present in 90% of the population, then 47% of cases would be expected to show evidence of memory. This analysis shows that memory can be quite efficacious in the population while at the same time failing in a subset of individuals. The calculated percentage range of Hib disease cases presenting with evidence of memory from these contingencies, 47 and 69%, does not differ significantly from the data of Anderson et al. when the 95% confidence intervals are taken into account (12 of 20 patients ≥18 months of age had evidence of memory, i.e. 60%; 95% confidence interval, 36 to 81%).
Thus we believe that the evidence presented in the study of Anderson et al. is insufficient to reject the idea that natural priming and the ability to evoke a memory antibody response to infection play an important role in protection against developing Hib disease. Furthermore conjugate vaccine-induced priming is likely to be qualitatively and quantitatively superior to natural priming. Conjugate vaccines prime for a higher magnitude memory antibody response 15, 37 and higher functional activity as a result of higher avidity antibody. 16
As reviewed recently, several lines of evidence support the idea that memory plays an essential role in Hib vaccine efficacy. 17 Immunization of Finnish infants at 3, 4 and 6 months, or 4 and 6 months, with a poorly immunogenic Hib conjugate vaccine, Hib PS-diphtheria toxoid, nonetheless resulted in high efficacy in preventing invasive Hib disease. 38–40 The degree of efficacy observed in those trials (90 and 87%, respectively) exceeded the percentage of infants who achieved antibody concentrations at 7 months of age considered sufficient to confer protection (40 and 32%, respectively, ≥1 μg/ml; and 68 and 73%, respectively, ≥0.15 μg/ml). Subsequently the Hib PS-diphtheria toxoid conjugate vaccine was found to prime for robust recall antibody responses to plain Hib PS vaccination. 41 Further evidence supporting a role for vaccine-induced immunologic priming in contributing to protection against Hib disease also comes from the postlicensure experience in the United Kingdom where Hib immunization is given to infants at 2, 3 and 4 months of age without a booster injection in the second year. Consequently by 1 year of age, 38% of UK infants have serum antibody concentrations below the 0.15 μg/ml threshold of protection, 19 yet vaccine efficacy in children >1 year of age in the UK remains at 94%. 42 The experience in the United States with use of plain Hib PS vaccination in children 2 to 5 years of age also is informative. This vaccine was immunogenic in this age group with the majority of those vaccinated developing ≥1 μg/ml serum antibody 43, 44 and with serum antibody concentrations being maintained at ≥0.15 μg/m in most individuals. 18 These antibody concentrations are higher than those in 2-year-olds who are currently being immunized as infants in the UK. However, vaccination with plain Hib PS vaccine does not prime memory antibody responses. 18 Between the interval when Hib PS vaccine was licensed in the US and when the vaccine was largely replaced by Hib conjugate vaccine, there were many reports of children developing Hib disease despite being vaccinated with plain Hib PS, 45, 46 there was no efficacy in some parts of the country, 47 and there was minimal evidence of a decline in the incidence of Hib disease in the age group being immunized with plain Hib PS vaccine (reviewed in Reference 48). In contrast after introduction of Hib conjugate vaccine, vaccine failure became rare and the incidence of Hib disease declined rapidly. 49, 50 Collectively these observations support the idea that a Hib vaccine that elicits serum antibody responses but induces poor memory is not nearly as effective in preventing disease as a vaccine that elicits both serum antibody and memory.
In summary we think a compelling case can be made that immunologic memory functions as a critical component of protective immunity to Hib. Memory, like antibody production, is imperfect, and it is unreasonable to expect otherwise. What is needed in this field is a balanced consideration of the relative roles of memory and humoral antibody in protection afforded by either natural exposure or vaccination. The current debate of whether antibody or memory is responsible for protection against Hib disease is reminiscent of the controversy between the humoralists and cellulists nearly 100 years ago. 51 Investigators could benefit by keeping this parallel in mind as they try to discern the relative contributions of these two interconnected aspects of acquired immunity to Hib disease. These considerations are not limited to Hib but are relevant to immunity to pneumococcal and meningococcal diseases for which new multivalent conjugate vaccines are being introduced.
This work was supported by National Institute of Allergy and Infectious Diseases Grants AI25008 and RR01271 (AHL) and AI45642 and AI46464 (DMG) from the National Institutes of Health.
1. Anderson P, Johnston RB Jr, Smith DH. Human serum activities against Hemophilus influenzae
, type b. J Clin Invest 1972; 51: 31–8.
2. Schneerson R, Rodrigues LP, Parke JC Jr, Robbins JB. Immunity to disease caused by Hemophilus influenzae
, type b: II. Specificity and biologic characteristics of “natural,” infection-acquired, and immunization-induced antibodies to the capsular polysaccharide of Hemophilus influenzae
, type b. J Immunol 1971; 107: 1081–9.
3. Schneerson R, Bradshaw M, Whisnant JK, Myerowitz RL, Parke JC Jr, Robbins JB. An Escherichia coli
antigen cross-reactive with the capsular polysaccharide of Haemophilus influenzae
type b: occurrence among known serotypes, and immunochemical and biologic properties of E. coli
antisera toward H. influenzae
type b. J Immunol 1972; 108: 1551–62.
4. Schneerson R, Robbins JB. Induction of serum Haemophilus influenzae
type B capsular antibodies in adult volunteers fed cross-reacting Escherichia coli
075: K100:H5. N Engl J Med 1975; 292: 1093–6.
5. Robbins JB, Schneerson R, Glode MP, et al. Cross-reactive antigens and immunity to diseases caused by encapsulated bacteria. J Allergy Clin Immunol 1975; 56: 141–51.
6. Anderson P. The protective level of serum antibodies to the capsular polysaccharide of Haemophilus influenzae
type b. J Infect Dis 1984; 149: 1034–5.
7. Käyhty H, Peltola H, Karanko V, Mäkelä PH. The protective level of serum antibodies to the capsular polysaccharide of Haemophilus influenzae
type b. J Infect Dis 1983; 147: 1100.
8. Käyhty H. Difficulties in establishing a serological correlate of protection after immunization with Haemophilus influenzae
conjugate vaccines. Biologicals 1994; 22: 397–402.
9. Käyhty H. Immunogenicity assays and surrogate markers to predict vaccine efficacy. Dev Biol Stand 1998; 95: 175–80.
10. Granoff DM, Lucas AH. Laboratory correlates of protection against Haemophilus influenzae
type b disease: importance of assessment of antibody avidity and immunologic memory
. Ann NY Acad Sci 1995; 754: 278–88.
11. Griswold WR, Lucas AH, Bastian JF, Garcia G. Functional affinity of antibody to the Haemophilus influenzae
type b polysaccharide. J Infect Dis 1989; 159: 1083–7.
12. Amir J, Liang X, Granoff DM. Variability in the functional activity of vaccine-induced antibody to Haemophilus influenzae
type b. Pediatr Res 1990; 27: 358–64.
13. Schlesinger Y, Granoff DM, Group TVS. Avidity and bactericidal activity of antibody elicited by different Haemophilus influenzae
type b conjugate vaccines. JAMA 1992; 267: 1489–94.
14. Insel RA, Anderson PW. Oligosaccharide-protein conjugate vaccines induce and prime for oligoclonal IgG antibody responses to the Haemophilus influenzae
b capsular polysaccharide in human infants. J Exp Med 1986; 163: 262–9.
15. Weinberg GA, Einhorn MS, Lenoir AA, Granoff PD, Granoff DM. Immunologic priming to capsular polysaccharide in infants immunized with Haemophilus influenzae
type b polysaccharide-Neisseria meningitidis
outer membrane protein conjugate vaccine. J Pediatr 1987; 111: 22–7.
16. Goldblatt D, Vaz AR, Miller E. Antibody avidity as a surrogate marker of successful priming by Haemophilus influenzae
type b conjugate vaccines following infant immunization. J Infect Dis 1998; 177: 1112–15.
17. Eskola J, Ward J, Dagan R, Goldblatt D, Zepp F, Siegrist CA. Combined vaccination of Haemophilus influenzae
type b conjugate and diphtheria-tetanus-pertussis containing acellular pertussis. Lancet 1999; 354: 2063–8.
18. Käyhty H, Karanko V, Peltola H, Mäkelä PH. Serum antibodies after vaccination with Haemophilus influenzae
type b capsular polysaccharide and responses to reimmunization: no evidence of immunologic tolerance or memory. Pediatrics 1984; 74: 857–65.
19. Goldblatt D, Miller E, McCloskey N, Cartwright K. Immunological response to conjugate vaccines in infants: follow up study. BMJ 1998; 316: 1570–1.
20. Goldblatt D, Richmond P, Millard E, Thornton C, Miller E. The induction of immunologic memory
after vaccination with Haemophilus influenzae
type b conjugate and acellular pertussis-containing diphtheria, tetanus, and pertussis vaccine combination. J Infect Dis 1999; 180: 538–41.
21. Zepp F, Schmitt HJ, Kaufhold A, et al. Evidence for induction of polysaccharide specific B-cell-memory in the 1st year of life: plain Haemophilus influenzae
type b-PRP (Hib) boosters children primed with a tetanus-conjugate Hib-DTPa-HBV combined vaccine. Eur J Pediatr 1997; 156: 18–24.
22. Pichichero ME, Passador S. Administration of combined diphtheria and tetanus toxoids and pertussis vaccine, hepatitis B vaccine, and Haemophilus influenzae
type b vaccine to infants and response to a booster dose of Hib conjugate vaccine. Clin Infect Dis 1997; 25: 1378–84.
23. Anderson P, Ingram DL, Pichichero ME, Peter G. A high degree of natural immunologic priming to the capsular polysaccharide may not prevent Haemophilus influenzae
type b meningitis. Pediatr Infect Dis J 2000; 19: 589–91.
24. O’Reilly RJ, Anderson P, Ingram DL, Peter G, Smith DH. Circulating polyribophosphate in Hemophilus influenzae
, type b meningitis: correlation with clinical course and antibody response. J Clin Invest 1975; 56: 1012–22.
25. Norden CW, Michaels RH, Melish M. Serologic responses of children with meningitis due to Haemophilus influenzae
type b. J Infect Dis 1976; 134: 495–9.
26. Granoff DM, Congeni B, Baker R Jr, Ogra P, Nankervis GA. Countercurrent immunoelectrophoresis in the diagnosis of Haemophilus influenzae
type b infection: relationship of detection of capsular antigen to age, antibody response, and therapy. Am J Dis Child 1977; 131: 1357–62.
27. Murphy TV, Osterholm MT, Pierson LM, et al. Prospective surveillance of Haemophilus influenzae
type b disease in Dallas County, Texas, and in Minnesota. Pediatrics 1987; 79: 173–80.
28. Robbins JB, Schneerson R, Anderson P, Smith DH. The 1996 Albert Lasker Medical Research Awards: Prevention of systemic infections, especially meningitis, caused by Haemophilus influenzae
type b: impact on public health and implications for other polysaccharide-based vaccines. JAMA 1996; 276: 1181–5.
29. Robbins JB, Parke JC Jr, Schneerson R, Whisnant JK. Quantitative measurement of “natural” and immunization-induced Haemophilus influenzae
type b capsular polysaccharide antibodies. Pediatr Res 1973; 7: 103–10.
30. Gigliotti F, Insel RA. Protection from infection with Haemophilus influenzae
type b by monoclonal antibody to the capsule. J Infect Dis 1982; 146: 249–54.
31. Lucas AH, Granoff DM. Functional differences in idiotypically defined IgG1 anti- polysaccharide antibodies elicited by vaccination with Haemophilus influenzae
type b polysaccharide-protein conjugates. J Immunol 1995; 154: 4195–202.
32. Santosham M, Rivin B, Wolff M, et al. Prevention of Haemophilus influenzae
type b infections in Apache and Navajo children. J Infect Dis 1992; 165 (Suppl 1): S144–51.
33. Barington T, Hougs L, Juul L, et al. The progeny of a single virgin B cell predominates the human recall B cell response to the capsular polysaccharide of Haemophilus influenzae
type b. J Immunol 1996; 157: 4016–27.
34. Hougs L, Juul L, Ditzel HJ, Heilmann C, Svejgaard A, Barington T. The first dose of a Haemophilus influenzae
type b conjugate vaccine reactivates memory B cells: evidence for extensive clonal selection, intraclonal affinity maturation, and multiple isotype switches to IgA2. J Immunol 1999; 162: 224–37.
35. Baxendale HE, Davis Z, White HN, Spellerberg MB, Stevenson FK, Goldblatt D. Immunogenetic analysis of the immune response to pneumococcal polysaccharide. Eur J Immunol 2000; 30: 1214–23.
36. Lucas AH, Moulton KD, Tang VR, Reason DC. Combinatorial library cloning of human antibodies to Streptococcus pneumoniae
capsular polysaccharides: variable region primary structures and evidence for somatic mutation of Fab fragments specific for capsular serotypes 6B, 14 and 23F. Infect Immun 2001; 69: 853–64.
37. Granoff DM, Holmes SJ, Osterholm MT, et al. Induction of immunologic memory
in infants primed with Haemophilus influenzae
type b conjugate vaccines. J Infect Dis 1993; 168: 663–71.
38. Eskola J, Käyhty H, Takala AK, et al. A randomized, prospective field trial of a conjugate vaccine in the protection of infants and young children against invasive Haemophilus influenzae
type b disease. N Engl J Med 1990; 323: 1381–7.
39. Eskola J, Peltola H, Käyhty H, Takala AK, Mäkelä PH. Finnish efficacy trials with Haemophilus influenzae
type b vaccines. J Infect Dis 1992; 165 (Suppl 1): S137–8.
40. Peltola H, Kilpi T, Anttila M. Rapid disappearance of Haemophilus influenzae
type b meningitis after routine childhood immunisation with conjugate vaccines. Lancet 1992; 340: 592–4.
41. Käyhty H, Eskola J, Peltola H, Saarinen L, Mäkelä PH. High antibody responses to booster doses of either Haemophilus influenzae
capsular polysaccharide or conjugate vaccine after primary immunization with conjugate vaccines. J Infect Dis 1992; 165 (Suppl 1): S165–6.
42. Booy R, Heath PT, Slack MP, Begg N, Moxon ER. Vaccine failures after primary immunisation with Haemophilus influenzae
type-b conjugate vaccine without booster. Lancet 1997; 349: 1197–202.
43. Granoff DM, Munson RS Jr. Prospects for prevention of Haemophilus influenzae
type b disease by immunization. J Infect Dis 1986; 153: 448–61.
44. Peltola H, Käyhty H, Virtanen M, Mäkelä PH. Prevention of Hemophilus influenzae
type b bacteremic infections with the capsular polysaccharide vaccine. N Engl J Med 1984; 310: 1561–6.
45. Granoff DM, Sheetz K, Pandey JP, et al. Host and bacterial factors associated with Haemophilus influenzae
type b disease in Minnesota children vaccinated with type b polysaccharide vaccine. J Infect Dis 1989; 159: 908–16.
46. Granoff DM, Shackelford PG, Suarez BK, et al. Hemophilus influenzae
type B disease in children vaccinated with type B polysaccharide vaccine. N Engl J Med 1986; 315: 1584–90.
47. Osterholm MT, Rambeck JH, White KE, et al. Lack of efficacy of Haemophilus
b polysaccharide vaccine in Minnesota. JAMA 1988; 260: 1423–8.
48. Murphy TV. Vaccines for Haemophilus influenzae
type b. Semin Pediatr Infect Dis 1991; 2: 120–34.
49. Murphy TV, White KE, Pastor P, et al. Declining incidence of Haemophilus influenzae
type b disease since introduction of vaccination. JAMA 1993; 269: 246–8.
50. Adams WG, Deaver KA, Cochi SL, et al. Decline of childhood Haemophilus influenzae
type b (Hib) disease in the Hib vaccine era. JAMA 1993; 269: 221–6.
51. Tauber A, Chernyak L. Metchnikoff and the origins of immunology: from metaphor to theory. Oxford, UK: Oxford University Press, 1991.