RESEARCH

Complement and Immunity to Pneumococcal Vaccines

Figure 1.  Structure of Streptococcus pneumoniae (reproduced with permission of The National Foundation for Infectious Diseases)

We have been studying the role of complement in immunity to vaccines against the capsular polysaccharides of Streptococcuspneumoniae, the pneumococcus.  Pneumococcal infections are a major public health concern throughout the world, with the very young, the elderly, and immunocompromised individuals being particularly susceptible to infection.  In the U.S., S. pneumoniae causes more cases of meningitis and pneumonia than any other species of bacteria.  Worldwide, over one million children die annually from pneumococcal infections.  There are over 90 different strains (serotypes) of the pneumococcus based on differences in structure of the capsular polysaccharide and the antibodies produced against each one are different. 

Pneumococcal vaccines must therefore contain polysaccharides from several different strains to protect against infection.  The original pneumococcal vaccines contained the capsular polysaccharides of 23 different pneumococcal serotypes.  For our research we have been using the polysaccharide from serotype 14 pneumococcus, a strain that frequently causes infections, often severe ones.  The capsular polysaccharide of type 14 pneumococcus (PPS14) consists of up to a thousand copies of a 4-sugar–long subunit.  Figure 2 shows a model of the subunit (left) and of a chain of 4 subunits linked together (right).

Figure 2.  Chemical structure of the serotype 14 pneumococcal capsular polysaccharide subunit (left) and of a 4-subunit chain (right)

Conjugation of C3d to serotype 14 pneumococcal capsular polysaccharide enhances the anti-polysaccharide antibody response.

Based on the results of others demonstrating that coupling several molecules of C3d to protein antigens caused a large increase in the amount of antibody produced by immunized mice, we prepared vaccines in which multiple copies of C3d were attached to type 14 pneumococcal capsular polysaccharide (PPS14).  We then immunized mice with two injections of vaccine and measured the antibody response.  Figure 3 shows that compared with unmodified PPS14, PPS14 with C3d attached caused the production of about 20 times as much antibody.

Figure 3.  Comparison of serum anti-PPS14 antibody concentrations after immunization of mice with two subcutaneous injections of PPS14 or PPS14-C3d.

We also found that immunization with two doses of PPS14-C3d could induce an antibody response comparable to that induced by two injections of a conjugate of PPS14 and ovalbumin (OVA), a T-dependent protein carrier.  Immunization with either conjugate induced anti-PPS14 antibodies primarily of the IgG1 isotype.  Immunizations in nude mice, which are deficient in T cells, showed that the primary antibody response to PPS14-OVA was significantly diminished in the absence of T cells, while the response to PPS14-C3d was only minimally affected.  However, there was no boost in antibody concentrations after secondary immunization with either PPS14-C3d or PPS14-OVA.

Reference:  Test ST, Mitsuyoshi J, Connolly CC, Lucas AH.  Increased immunogenicity and induction of class switching by conjugation of complement C3d to pneumococcal serotype 14 capsular polysaccharide.  Infect Immun 69:3031-3040, 2001.

Subsequent to our original studies on PPS14-C3d conjugates, we found that they did not consistently induce antibody responses that were as strong as those induced by PPS14-OVA conjugates.  We also found that PPS14-OVA conjugates induced a greater switch in anti-PPS14 antibodies from the IgM to the IgG subclass than did PPS14-C3d conjugates.  We were therefore curious whether the IgG antibodies induced by the two different conjugate vaccines were functionally similar.  IgG antibodies were purified from serum samples obtained from mice at 25 days after secondary immunization and compared in assays of antibody avidity, opsonophagocytosis of serotype 14 pneumococci by murine RAW 264.7 macrophages, and killing of serotype 14 pneumococci by RAW 264.7 cells.  Compared with anti-PPS14 IgG induced by immunization with PPS14-C3d, IgG antibodies induced after immunization with PPS14-OVA were of higher avidity, had higher opsonophagocytic activity, and resulted in killing of greater numbers of bacteria.  Thus, although PPS14-C3d conjugate vaccines are more effective than unmodifed PPS14, they appear to be inferior to PPS14 conjugates incorporating protein carriers, both in terms of the magnitude of the anti-PPS14 antibody response induced and in the functional attributes of the IgG anti-PPS14 antibodies.  For these reasons, they are unlikely to replace the currently used conjugate vaccines in clinical practice.  Nonetheless, they remain extremely valuable tools for assessing the role of C3d-CR2 intreractions in the immune response to PPS14.

Reference: Hu Y, Test ST.  Functional differences in IgG anti-polysaccharide antibodies elicited by immunization of mice with C3d versus ovalbumin conjugates of pneumococcal serotype 14 capsular polysaccharide.  Vaccine 23:21-28, 2004.

Depletion of complement inhibits the primary but enhances the secondary anti-PPS14 antibody response after immunization with PPS14-OVA conjugates.

Administration of vaccines often involves a series of several injections given over months or years.  The antibody response to the second or later injection differs from the response to the first injection and is called a secondary or recall response.  The secondary antibody response occurs more rapidly than the primary response, consists of more IgG antibody (the primary response consists primarily of IgM antibody), and consists of antibodies that bind more strongly to the antigen.

Figure 4.  Comparison of serum anti-PPS14 IgM and IgG concentrations at 10 days after primary (left) or secondary (right) immunization with 0.2 µg PPS14-OVA in untreated mice or mice treated with cobra venom factor at the time of primary immunization to deplete serum complement (C3).

We observed similar effects at PPS14-OVA doses of 0.5 and 1 µg and found that the effect was maximal when complement was depleted within 2 days of primary immunization.  IgG1 anti-PPS14 predominated in both untreated and cobra venom factor–treated mice.  Treatment of mice with cobra venom factor also had no effect on the functional activity of IgG purifed from 25 day post-secondary serum samples.  Overall, these data suggest that complement activation at the time of primary immunization favors the primary antibody response at the expense of the secondary response.  This could occur by a number of different mechanisms, including targeting of antigen to selected populations of immune cells or favoring the differentitation of antigen-specific B cells into antibody-secreting plasma cells instead of memory B cells.

Because pneumococcal conjugate vaccines are difficult to manufacture, shortages have already occurred.  We believe that administering complement inhibitors at the time of the first injection may be a way to reduce vaccine usage, by allowing a reduction in either dosage or in the number of injections required to produce sufficient amounts of antibody to protect against infection.

The goal of our current research is to understand the cellular and molecular mechanisms by which complement affects the primary and secondary antibody responses to polysaccharide vaccines and to determine whether manipulations of the complement system can be used to improve the antibody response to other types of vaccines as well.

Reference: Test ST, Mitsuyoshi JK, Hu Y.  Depletion of complement has distinct effects on the primary and secondary antibody responses to a conjugate of pneumococcal serotype 14 capsular polysaccharide and a T-cell-dependent protein carrier.  Infect Immun 73:277-286, 2005.