pneumococcal conjugate vaccine (PCV or Prevnar-13
® ) contains
polysaccharides from 13 serotypes covalently linked to a nonpathogenic diphtheria toxoid that triggers a T-cell dependent antibody
response [3]. The pneumococcal capsule is known to inhibit
phagocytosis by immune cells [1]; therefore, one way antibodies
induced following administration of vaccines can protect the host is
by opsonizing S. pneumoniae and facilitating their uptake and
killing by host cells [5].
Vaccine responses are typically assessed by measuring the levels
of serotype-specific anticapsular antibodies against pneumococci
using standard enzyme-linked immunosorbent assays (ELISA)
[6]. However, antibody levels are not always indicative of vaccine
efficacy since they do not reflect functionality, defined as the ability
to opsonize and enhance phagocytic uptake of bacteria [6]. For
example, in vulnerable populations, such as the elderly, postvaccination sera had significantly reduced opsonophagocytic activity
against S. pneumoniae [7, 8] when compared to young adults
despite the two populations having similar anti-polysaccharide antibody levels. Therefore, the ability of immune sera to act as an
efficient opsonin has routinely been determined with in vitro opsonophagocytosis assays using phagocytic cell lines such as HL-60
cells [9]. While a great tool for directly assessing antibody function,
these cell lines are not the ones mediating bacterial killing in vivo.
One of the most important defense mechanisms against pneumococci is polymorphonuclear cells (PMNs) also known as neutrophils. These cells rapidly migrate to sites of infection and are
required for host defense [10]. Neutropenic individuals or mice
predepleted for neutrophils are highly susceptible to IPD [10–13].
PMNs are viewed as effectors of vaccine responses. In other words,
vaccination triggers antibodies and one of the ways antibodies
protect the host against infection is by binding pneumococci and
promoting their clearance via enhancing uptake and killing by
PMNs [5]. There is evidence that age-related decline in the intrinsic
PMN function may contribute to impaired PPSV efficacy in the
elderly. Pneumococci that were opsonized with sera from young
PPSV immunized donors were killed less efficiently by PMNs from
elderly donors than by young controls suggesting that
Ab-mediated opsonophagocytic killing of S. pneumoniae by
PMNs is impaired in the elderly [5]. Therefore examining both
antibody as well as PMN function would give a more complete
assessment of vaccine efficacy in a given host.
The mouse model has extensively been used to demonstrate the
immunogenicity and protective efficacy of S. pneumoniae vaccines
in vivo [14, 15]. However, with the different serotypes present in
the vaccine formulations and the possibility of variation in antibody
and phagocytic cell responses, in vitro assays for measuring vaccine
responses are more economical and are needed for dissection of
mechanisms. Here we describe a one-stop protocol for in vitro
560
Manmeet Bhalla et al.
® ) contains
polysaccharides from 13 serotypes covalently linked to a nonpathogenic diphtheria toxoid that triggers a T-cell dependent antibody
response [3]. The pneumococcal capsule is known to inhibit
phagocytosis by immune cells [1]; therefore, one way antibodies
induced following administration of vaccines can protect the host is
by opsonizing S. pneumoniae and facilitating their uptake and
killing by host cells [5].
Vaccine responses are typically assessed by measuring the levels
of serotype-specific anticapsular antibodies against pneumococci
using standard enzyme-linked immunosorbent assays (ELISA)
[6]. However, antibody levels are not always indicative of vaccine
efficacy since they do not reflect functionality, defined as the ability
to opsonize and enhance phagocytic uptake of bacteria [6]. For
example, in vulnerable populations, such as the elderly, postvaccination sera had significantly reduced opsonophagocytic activity
against S. pneumoniae [7, 8] when compared to young adults
despite the two populations having similar anti-polysaccharide antibody levels. Therefore, the ability of immune sera to act as an
efficient opsonin has routinely been determined with in vitro opsonophagocytosis assays using phagocytic cell lines such as HL-60
cells [9]. While a great tool for directly assessing antibody function,
these cell lines are not the ones mediating bacterial killing in vivo.
One of the most important defense mechanisms against pneumococci is polymorphonuclear cells (PMNs) also known as neutrophils. These cells rapidly migrate to sites of infection and are
required for host defense [10]. Neutropenic individuals or mice
predepleted for neutrophils are highly susceptible to IPD [10–13].
PMNs are viewed as effectors of vaccine responses. In other words,
vaccination triggers antibodies and one of the ways antibodies
protect the host against infection is by binding pneumococci and
promoting their clearance via enhancing uptake and killing by
PMNs [5]. There is evidence that age-related decline in the intrinsic
PMN function may contribute to impaired PPSV efficacy in the
elderly. Pneumococci that were opsonized with sera from young
PPSV immunized donors were killed less efficiently by PMNs from
elderly donors than by young controls suggesting that
Ab-mediated opsonophagocytic killing of S. pneumoniae by
PMNs is impaired in the elderly [5]. Therefore examining both
antibody as well as PMN function would give a more complete
assessment of vaccine efficacy in a given host.
The mouse model has extensively been used to demonstrate the
immunogenicity and protective efficacy of S. pneumoniae vaccines
in vivo [14, 15]. However, with the different serotypes present in
the vaccine formulations and the possibility of variation in antibody
and phagocytic cell responses, in vitro assays for measuring vaccine
responses are more economical and are needed for dissection of
mechanisms. Here we describe a one-stop protocol for in vitro
560
Manmeet Bhalla et al.