687Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 99(7): 687-689, November 2004
Naturally Acquired Immunity to Haemophilus influenzae Type B in
Healthy Cuban Children
Gilda Toraño Peraza+, Ibis Hernández Vadell, María Eugenia Toledo Romaní,
Alberto Baly Gil, Isis Tamargo Martínez, Annia Carmenate García
National Reference Center for Haemophilus, Instituto de Medicina Tropical “Pedro Kourí”, Havana, Cuba
We have evaluated the prevalence of antibody to immunogenicity of Haemophilus influenzae type b (Hib) in a
group of 4 to 5 years old healthy children, who were too old to be included in the first vaccinated cohort when Hib
vaccination begun in Cuba in 1999. Serum capsular polysaccharide specific IgG antibody concentrations were
measured in 974 healthy children, between February and May 2002. The prevalence of Hib nasopharyngeal car-
riage was also estimated. The majority of children (99.7%) had more than 1 µg/ml of antibody. The preliminary
report of the nasopharyngeal cultures was positive for H. influenzae in 16 children, but in only one was confirmed as
Hib after serotyping (0.1% Hib nasopharyngeal carrier). These results provide evidence that in Cuba the natural
active immunity to Hib can be acquired at an early age.
Key words: Haemophilus influenzae type b - natural immunity - pre-vaccination antibody titres - Cuba
Haemophilus influenzae type b (Hib) disease is now a
major cause of vaccine-preventable morbidity and mor-
tality in young children in developing countries. Despite
the availability of a protective vaccine, few developing
countries are using Hib vaccine in their immunization pro-
grams. The major obstacle to the routine use of Hib con-
jugate vaccine in most non-industrialized countries is cost
(Fernández et al. 2000).
In Cuba the Hib vaccination was introduced in the
National Immunization Program in 1999, for all the chil-
dren born between January 1998 and October 1999. After
that, the incidence of Hib disease has decreased to 0.1 per
100,000 inhabitants, in 2001 (Dickinson et al. 2001). The
purpose of this study was to evaluate the natural acquired
immunity to Hib in a group of healthy children 4 to 5 years
of age in the last cohort not vaccinated against Hib.
This study was performed between February and May
2002, in the province of Camagüey, Cuba. Nine hundred
and seventy four healthy children attending day care cen-
ters and schools were selected. No child had previously
received Hib vaccine or had received any immunoglobu-
lin preparation or blood product. Demographic and medi-
cal data were obtained through a personal interview with
the mothers. Blood samples were drawn from children,
and the serum was stored at –20°C until further analysis.
Serum capsular polysaccharide specific IgG antibody
(anti-PRP) concentrations were measured by a modifica-
tion of the enzyme-linked immunosorbent assay (ELISA)
using HbOHA (NIBSC, Potters Bar, UK) as antigen. A stan-
+Correponding author. Fax: +53-7-204.6051. E-mail:
Received 8 April 2004
Accepted 17 September 2004
dard curve was generated by using reference serum (lot
1983 Center for Biological Evaluation and Review, Food
and Drug Administration, Bethesda, MD) with a calcu-
lated IgG antibody concentration (60.9 µg/ml) (Keythy et
al. 1987, Phipps et al. 1990). IgG antibody concentration
was logarithmic transformed and the geometric mean con-
centration (GMC) was calculated.
Nasopharyngeal swabs were collected and inoculated
on chocolate agar medium supplemented with V and X
factor, and incubated in 5% CO2 in air at 37°C overnight.
Isolates were identified as H. influenzae by their require-
ments for V and X factors (BBL TAXO X and V FACTOR
STRIPS, Becton Dickinson Microbiology System).
Serotyping of isolated strains was performed by slide
agglutination with specific antisera for the capsular types
a to f (Difco Laboratories, Detroit, MI).
The results of anti-PRP specific IgG antibody studied
in Cuban children aged 4 to 5 years are shown in the
Table. Nine hundred and seventy two children (99.7%)
had protective level of antibodies. The greatest percent-
age of children (59.75% and 26.07%) corresponded to pro-
tective titres of antibodies between 1-5 µg/ml and 5 µg/ml,
respectively. Titres expected to be protective for immedi-
ate but short-term periods (> 0.15 and < 1 µg/ml) were
observed in 136 children (13.96%). The GMC was 2.71
The preliminary report of the nasopharyngeal cultures
was positive for H. influenzae in 16 children, but the iden-
tification was confirmed by serotyping in only one cul-
ture (0.1% Hib nasopharyngeal carrier). The rest of the
isolates were noncapsulated, serologically nontypeable
In the era prior to Hib vaccination in Finland, research
has shown that the majority of children 4 to 5 years of age
had protective concentrations of antibody. Kaythy et al.
(1991, 1992) reported that 79% had titres higher than 0.15
µg/ml and 32% had titres greater than 1 µg/ml. Similar
688Natural Immunity to H. influenzae b in Cuba • Gilda Toraño Peraza et al.
findings were noted in India, demonstrating high pre-vac-
cination anti-PRP titres in over 80% of children over 4
years of age (Acharya et al. 1997). The GMC in both stud-
ies (0.48 µg/ml in Finland and 0.94 µg/ml in India) was
lower than GMC obtained in this research (2.71 µg/ml).
The high pre-vaccination antibody levels in Cuban
children suggest that the children had been primed by
natural infection. In the pre-vaccine era, most children
acquired “natural” immunity by 5-6 years of age through
asymptomatic infection by Hib bacteria. Since only a rela-
tively small proportion of children carry Hib at any time, it
has been postulated that exposure to organisms that share
common antigenic structures with the capsule of Hib (so-
called “cross-reacting organisms”) may also stimulate the
development of anticapsular antibodies against Hib
(Bradshaw et al. 1971). For example, biologically active
anti-PRP antibodies can be induced by colonization with
Escherichia coli K-100, other enteric bacteria, and also
Staphylococcus aureus. E. coli are ubiquitous in devel-
oping countries like Cuba, and their presence in the gut
may have helped to stimulate antibody to Hib (Scheerson
et al. 1995, Puliyel et al. 2001).
On the other hand, besides the prevention of invasive
infections, Hib conjugate vaccines reduce the oro/na-
sopharyngeal carriage of Hib because they induce the
development of mucosal antibodies than can prevent colo-
nization (Kaythy 1998). The transmission of Hib is de-
creased, and even those children not immunized will be
protected. This phenomenon could explain the finding of
only one Hib nasopharyngeal carrier was found among
the children enrolled in the trial. Another possible expla-
nation for this phenomenon is the finding that a large
number of children (26.07%) developed anti-PRP titres
even higher than 5 µg/ml. The serological correlations of
protection from Hib carriage are not well understood how-
ever, a high serum antibody concentration seems to be
needed to prevent colonization, higher than the concen-
tration needed for preventing invasive disease (Takala et
This study shows that the Cuban children had high
anti-PRP concentrations and provides strong evidence
that children in some developing countries acquire natu-
ral active immunity to Hib at an early age (Tastan et al.
2000, Puliyel et al. 2001, Clemens et al. 2003). These re-
sults support the recommendation of the World Health
Organization that the strategy of administering Hib vac-
cine to all children under 5 years old must be considered
only in those countries which can afforded it without to
diverting resources from essential infants vaccination
programs (WHO 1998).
Natural immunity to Hib seems to be different among
children from one country to another, depending on geo-
graphical, genetic, and social factors. The high natural
immunity may be accounted for by high endemicity of
Hib in some countries, and over time, it is possible that
the ability to mount an elevated response against the bac-
teria could be selected in the populations. We suggest
that each country should investigate the natural immu-
nity in children between 1-5 years of age, before deciding
whether to introduce Hib vaccine in routine immunization
programmes in children in this age group.
Acharya D, Bhave S, Joshi V, Bavdekar A, Pandit A 1997.
Haemophilus influenzae type b in India: need and timing,
immunogenicity and tolerance. Indian Pediatr 34: 9-15.
Bradshaw M, Scheerson R, Parke J, Robbins J 1971 Bacterial
antigens cross reactive with capsular polysaccharide of
Haemophilus influenzae type b. Lancet 1: 1095-1096.
Clemens S, Azevedo T, Homma A 2003. Feasibility study of
the immunogenicity and safety of a novel DTPw/Hib (PRP-
T) Brazilian combination compared to a licensed vaccine in
healthy children at 2, 4, and 6 months of age. Rev Soc Bras
Med Trop 36: 321-330.
Dickinson F, Pérez A, Galindo M, Quintana I 2001. Impact of
vaccination against Haemophilus influenzae type b in Cuba.
Rev Panam Salud Publica 10: 169-173.
Fernández J, Balter S, Feris J, Gómez E, Garib Z, Castellanos
P, Sánchez J 2000. Randomized trial of the immunogenic-
ity of fraccional dose regimens of PRP-T Haemophilus
influenzae type b conjugate vaccine. Am J Trop Med Hyg
Kayhty H 1998. Immunogenicity assay and surrogate markers
to predict vaccine efficacy. In F Brown, S Plotkin (eds),
Preclinical and Clinical Development of New Vaccines, Dev
Biol Stand Basel, Karger, Vol. 95, p. 175-180.
Kaythy H, Eskola J, Peltola H, Stout M, Samuelson J, Gordon
L 1987. Immunogenicity in infants of a vaccine composed
of Haemophilus influenzae type b capsular polysaccharide
mixed DPT or conjugated to diphtheria toxoid. J Infect Dis
Kaythy H, Eskola J, Peltola H, Ronnberg P, Kela E, Keranko V,
Saarinen L 1991. Antibody response to four Haemophilus
influenzae type b conjugate vaccines. Am J Dis Child 145:
Kaythy H, Eskola J, Peltola H, Saarinen L, Makela PH 1992.
High antibody responses to booster doses of either
Haemophilus influenzae type b capsular polysaccharide or
conjugated vaccine after primary immunization with conju-
gated vaccines. J Infect Dis 165 (Suppl. 1): S165-S166.
Phipps D, West J, Eby R, Koster M, Madore D, Kuataert S
1990. An ELISA employing a Haemophilus influenzae type
b oloigosaccharide; human serum albumin conjugate corre-
lates with the radioantigen binding assay. J Immunol Meth-
ods 135: 121-128.
Puliyel J, Agarwal K, Abass F 2001. Natural immunity to
Haemophilus influenzae type b in infancy in Indian chil-
dren. Vaccine 19: 4592-4594.
Schneerson R, Robbins JB 1995. Induction of serum
Haemophilus influenzae type b capsular antibodies in adult
Serum capsular polysaccharide specific IgG antibody (anti-
PRP) titres measured by ELISA in 4-5 years old Cuban
children no vaccinated against Haemophilus influenzae b
< 0.15 2
> 0.15 and < 1
> 1 and < 5
GMC: geometric mean concentration of IgG antibody; a: p <
0.05; confidence intervals: 2.54-2.78
689Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 99(7), November 2004
volunteers fed cross-reacting Escherichia coli 075: K100:H5.
New England J Med 292: 1093-1096.
Takala AK, Eskola J, Leimonen M, Kaythy H, Nissinen A,
Pekkanen E, Makela PH 1991. Reduction of oropharyngeal
carriage of Haemophilus influenzae type b (Hib) in children
immunized with an Hib conjugated vaccine. J Infect Dis
Tastan Y, Alikasifoglu M, Ilter O, Erginoz, Arvas A, Yuksal D,
Turkcu F, Badur S 2000. Natural immunity to Haemophilus
influenzae type b among healthy children in Istambul, Tur-
key. Indian Pediatrics 37: 414-417.
WHO 1998. Global Programme for Vaccines and Immunisation.
The WHO position paper on Haemophilus influenzae type
b conjugate vaccines. WER 73: 64-68.