# Abstract

The development and licensure of vaccines against encapsulated bacteria
based protein polysaccharide conjugates has greatly increased the
protection of infants and children against meningitis and otitis media.
Current conjugate vaccines while effective do not protect against the
carrier protein and are manufactured using a low efficiency random
coupling procedures. Ideally a well-defined vaccine is effective and
easy to manufacture and characterize consistently. We propose to develop
a procedure for the preparation of well defined polysaccharide --tetanus
"toxoid" using efficient chemistry in which the oligosaccharide chains
are targeted to specific non-epitope containing sites on a recombinant
toxoid carrier protein. If successful such a procedure could lead to the
development of vaccines that protect against more than one pathogen and
is straightforward to characterize thus reducing the regulatory burdens
due to manufacturing.

### Specific Aims

1.  To develop tetanus toxin fragments with residues suitable for
    coupling carbohydrate antigens.

2.  To develop efficient procedures for directional coupling of
    oligosaccharide to proteins.

3.  To test the ability of conjugates to induce antibody to
    oligosaccharide and tetanus antigens.

### Background

Although many different microorganisms may cause meningitis in neonates,
most invasive bacterial disease occurring after 2-months of age has
historically resulted from infection by one of three bacterial species:
Haemophilus influenzae, Streptococcus pneumoniae, or Neisseria
meningitidis. The polysaccharide capsules synthesized by these pathogens
are attractive targets for vaccine development because they are located
on the bacterial cell surface and function as primary virulence
determinants of disease. Currently, conjugated polysaccharide vaccines
against type b H. influenzae, 11 or more S. pneumoniae serotypes, and N.
meningitidis groups A, C, Y and W135 are proving their efficacy and
safety in preventing meningitis in children and young adults. A
polysaccharide conjugate vaccine against Staphylococcus aureus infection
also appears as if it will be clinically successful.

Although these conjugate vaccines are successful, the nature of the
components necessary for eliciting protective antibody is ill defined.
This situation is at least partly due to the random nature in which the
saccharides are coupled to the carrier proteins and thus an ambiguous
structure of the conjugate vaccine. Random coupling may also result in
modification of important protective epitomes on the carrier protein.
Although current conjugates are prepared from components to two
different organisms they are designed to protect against a single
pathogen. We suggest that a new generation of polysaccharide conjugate
vaccines would build on the successes of those already in use by
controlling the extent of polysaccharide valence and defining the sites
of attachment to the conjugated (polypeptide) partner. These rationally
designed vaccines would be tested for efficacy in an animal model,
conceivably leading to markedly improved immunogens.

A report of the National Vaccine Advisory Committee on strengthening the
supply of routinely recommended vaccines in the United States was
published in January 2003. The report cites cost, cGMP requirements and
problems in manufacturing as some of the factors that affect the vaccine
supply. The efficiency of the manufacturing process and ease with which
the consistency of the product is monitored affects both the cost of
production and the outcome of the regulatory review of the product. The
efficiency of coupling protein to carbohydrate antigens in currently
licensed vaccines is 10-25%. Development of efficient conjugation
procedures and well defined conjugate vaccines would have a positive
effect on the vaccine supply since they would reduce production cost and
make the characterization and review of manufacturing more
straightforward.

We propose to improve coupling efficiency and to define the limits of
polysaccharide conjugate vaccines by coupling oligosialic acid to
chemically defined derivatives of the tetanus toxin C fragment, thereby
allowing us to control the number and position of the polysaccharide
chains on the carrier polypeptide. Such vaccines will serve as a
platform for future investigation of the molecular details of
polysaccharide-protein conjugate immunity.

### Study design/Plan

Approach- To construct conjugate vaccines against meningococcus groups C
and E. coli K92 by coupling purified capsular oligosialic acids to
defined residues in the N-terminal domain of the tetanus toxin Hc
fragment. To test the immunogenicity of the conjugates in mice.Specific
Aim 1 -- To develop tetanus toxin fragments with residues suitable for
coupling carbohydrate antigens - The basic experimental protocol will be
to couple oligosialic acids with reagents specific for conjugation to
cysteine residues of the carrier protein, tetanus Hc fragment. The Hc
fragment is composed of an N-terminal P-lectin-like domain and a
C-terminal beta-trefoil domain that includes the structural information
for toxin binding to host cells. Because most of the host's immune
response to tetanus toxin is directed toward this binding domain, we
hypothesize that the effectiveness of conjugate vaccines would be
maximized by retention of the beta-trefoil and thereby potentially
provide immunity to both meningococcus and tetanus. Hc fragment will be
purified as an N-terminal fusion to a hexahistidine tag by metal-ion
chromatography. To avoid conjugation with the binding domain, which
might disrupt structure, cysteine residues in the beta-trefoil will be
altered to alanines by site-directed mutagenesis. On the basis of the
three-dimensional crystal structure of the Hc fragment, these cysteine
alterations are not predicted to affect structure. We will introduce
cysteine alterations into solvent-exposed turns of the lectin-like
domain resulting in Hc fragments with potential valence of 2 to 6
oligosialic acid chains per polypeptide, being careful not to disrupt
T-cell dependent epitopes of the carrier.Should the use of the directed
cysteine approach result in too many technical problems, alternative
tetanus Hc fragments will be developed to selectively attach
oligosaccharides to amino terminal serine or threonine residues. Amino
terminal serine and threonine residues at neutral pH are rapidly
oxidized to yield terminal aldehyde residues. These aldehydes can be
reductively aminated efficiently, thus providing a means of selectively
attaching an oligosaccharide to the amino terminus of the Hc
fragment.Specific Aim 2 - To develop efficient procedures for
directional coupling oligosaccharide to proteins. -- Meningococcal group
C is a major cause of bacterial meningitis. Meningococcal group C and E.
coli K92 polysaccharides are polysialic acids. Group C is an a(2-9)
polysialic acid and the K92 is an alternating a(2-8)a(2-9) polysialic
acid. We will take advantage of a recombinant a(2-8) specific
endoneuraminidase to prepare oligosaccharides. Because of the
availability of this convenient reagent and the similarity of the
polysaccharides K92 oligosaccharides will be used as a model for
developing coupling reactions. The objective of the experiment is to
couple the reducing end of the oligosaccharides to specific sites on Hc
thus creating a directionally coupled conjugate. The reducing ends of
polysialic acids are not very reactive. To improve the reactivity of
oligosialic acids and therefore coupling efficiency, amines or thiol
reactive maleimides will be introduced at the end of the
oligosaccharides using modifications of standard reductive amination
methodology. The efficiency of the reductive amination reaction will be
improved by the use of aprotic solvents and by the concentration and
structure of the amine nucleophile. Oligosaccharides containing terminal
maleimides will be conjugated to cysteine residues in tetanus Hc
fragment. Oligosaccharides in which terminal free amines were introduced
will be conjugated to aldehydes generated by periodate oxidation
aminoterminal 2-amino alcohols.Specific Aim 3 -- Immunogenicity of the
conjugate vaccines: To determine the relative effectiveness of valence
and positional substitution on immunogenicity of the different conjugate
vaccines.The expected results of Aims 1 and 2 are a series of conjugates
with various defined structures. The immunogenicity of these potential
vaccines will be tested in mice. Groups of BALB/c mice (three groups of
3 mice per conjugate or buffer control) will be immunized
sub-cutaneously with three doses (2.5 mg total sialic acid in 0.5 ml
PBS) of conjugate vaccine in adjuvant at two-week intervals. Mice will
be bled one week after each injection and IgG response measured by ELISA
using HRP-conjugated goat anti-mouse IgG. Plates will be coated with the
conjugate or Hc fragment and the anti-polysaccharide antibody titer
determined by difference. Specificity will be determined by
preadsorption with polysaccharide or Hc fragment.Expected OutcomesAt
completion of the proposed research we will have designed and tested a
series of chemically and structurally defined conjugate polysaccharide
vaccines. The proposed analysis of these vaccines will allow us to
determine the effect of conjugate valence on immunogenicity in a defined
animal model that has been shown to mimic the human response to
polysaccharide vaccines. In addition, we will have determined whether
the vaccine provides for a strong immune response to both the carrier
and polysaccharide conceivably resulting in an immunogen that is
protective to both the group C meningococcus and tetanus. Future
directions would be to extend these coupling procedures to other
polysaccharide conjugates containing other types of polysaccharide
structures.