Structural Biology and Pathogen Entry

Figure 2. Figure 2. Structure of Viral or Bacterial Glycoproteins and Their Role in Host Invasion. A detailed knowledge of the mechanism by which viral glycoproteins mediate entry into host cells can now be applied to pathogens that once were not susceptible to vaccines, including human immunodeficiency virus (HIV) (Panel A, Protein Data Bank code 3JWD), influenza virus (Panel B, Protein Data Bank code 1RU7), and meningococcus (Panel C, adapted with permission from Scarselli et al.; Protein Data Bank code 2Y7S).9-11MPER denotes membrane proximal external region, and V1V2 variable regions 1 and 2. The Protein Data Bank is accessible at www.pdb.org.

Progress in virology, genetics, synthetic biology, and biotechnology has provided a new set of tools to approach current-day vaccinology. Among currently licensed vaccines, the most consistent biomarker for vaccine efficacy has been the presence of antibodies that neutralize the pathogen. These antibodies are often elicited by natural infection or immunization. Our understanding of the molecular structure of viruses has led to a sophisticated understanding of viral glycoproteins and the specific interactions of antibodies that can inactivate them. The field of structural biology has provided new insights into how such antibodies protect against infection by poliomyelitis, measles, and influenza viruses, as well as human papillomavirus (HPV), among others. This detailed knowledge of the mechanism by which viral glycoproteins mediate entry into host cells can now be applied to pathogens that have not been susceptible to this therapeutic approach (Figure 2).9-11 Thus, an understanding of the steps related to entry and survival of pathogens that cause illnesses such as HIV type 1 (HIV-1) infection, tuberculosis, and malaria offers molecular targets that serve both to understand natural infection and to identify highly conserved and invariant structures as targets for broadly neutralizing antibodies.

Rational Vaccine Design

The definition of conserved sites of vulnerability on pathogens provides the basis for structure-based vaccine design. Broadly neutralizing antibodies often recognize highly conserved sites that are susceptible to antibody inactivation. Two pathogens, HIV-1 and influenza virus, have proved to be particularly informative in this regard. For example, analysis of the HIV-1 envelope has revealed at least four discrete sites that represent potential targets for the designs of immunogens (i.e., agents capable of inducing an immune response). These include the CD4-binding site, a glycosylated site in variable regions 1 and 2 (V1V2), glycans on the outer domain, and the membrane proximal external region.

Progress in HIV-vaccine research has been advanced recently by the identification of exceptionally broad and potent neutralizing antibodies to each of these sites. Some monoclonal antibodies neutralize more than 90% of circulating viral strains,12-17 creating new opportunities for HIV-vaccine development. Similar progress has been made in the identification of broadly neutralizing antibodies directed against diverse influenza viruses. At least two independent sites of vulnerability have been identified, one in the stem region of the viral spike that helps to stabilize the trimer, the three identical viral hemagglutinin glycoproteins that form this structure, and the other in the receptor-binding region that recognizes sialic acid.18 The existence of such antibodies provides conceptual support and tools that facilitate the development of universal influenza vaccines intended to protect against a wide array of viruses, not only the circulating seasonal strain.

Knowledge of atomic structure also defines viral proteins to elicit these broadly neutralizing antibodies. For HIV infection, alternative forms of envelope glycoproteins include trimers, monomers, subdomains, and specific peptide loops transplanted onto scaffolds.19 These candidate vaccines are further modified with the use of protein-design algorithms that are based on bioinformatics10 in efforts to stabilize the immunogen, better expose the conserved sites, and mask or remove undesired epitopes. Similar strategies are under development for influenza viruses, respiratory syncytial virus, and group B meningococcal strains.9,11,18,20,21

Although structure-based rational design offers a promising tool for developing vaccines against recalcitrant pathogens, substantial challenges remain. The proper antigenic structure will not necessarily provide all the information needed to produce a potent immunogen that will elicit an antibody response. Furthermore, many broadly neutralizing antibodies are atypical, with an unusually high degree of somatic mutation or long CDRH3 (third complementarity determining regions of heavy-chain variable) regions; such antibodies may not be readily elicited. Finally, a successful vaccine candidate must be designed to bind the germline antibody precursor, select for the appropriate primary recombinational events, and direct its somatic mutations toward the appropriate mature form.19

Interactions between Host and Pathogen

Figure 3. Figure 3. Molecular Evolution of a Successful Broadly Neutralizing Antibody. Deep sequencing (i.e., the ability to generate millions of independent sequences of a gene product) identifies critical intermediates for the evolution of broadly neutralizing antibodies and guides vaccine development. In Panel A, maximum-likelihood trees of heavy-chain sequences were derived from the IGHV1-2 gene that gives rise to a broadly neutralizing antibody, VRC01, in a representative patient, donor 74, as described previously.23 The donor 74 tree is rooted in the putative reverted unmutated ancestor of the heavy chain of a specific broadly neutralizing CD4-binding site monoclonal antibody, VRC-PG04 (as shown in Panel B, Protein Data Bank code 3SE9). Sequences from other donors are included in the cross-donor phylogenetic analysis. Bars representing 0.1 changes per nucleotide site are shown. Sequences within the shaded box include autologous VRC01-like heavy-chain sequences that neutralize HIV with good potency and breadth and are probably clonal relatives of VRC-PG04. Sequences highlighted in blue and purple represent broadly neutralizing antibodies isolated with structural probes.

Progress in the field of therapeutic monoclonal antibodies has facilitated the identification of effective targets and led to strategies for their successful use in humans.22 Dozens of new antibodies directed against HIV-1,18,19 influenza virus,21 respiratory syncytial virus,20 hepatitis C virus,18 and other microbes have identified critical viral structures and enabled structure-based vaccine design. Moreover, deep sequencing, the ability to generate millions of independent sequences of a gene product (e.g., immunoglobulin), has identified intermediates that are critical for the evolution of broadly neutralizing antibodies and has guided vaccine development.23 Millions of gene sequences encoding heavy and light chains (the polypeptide subunits of an antibody) within a single individual can be analyzed with the use of bioinformatics to trace a potential critical path for vaccine design (Figure 3).23 The overarching goal is to use knowledge of structural biology and antibody evolution to design vaccines that will elicit antibodies of known specificity.24

Genomewide sequencing of microbes has also allowed for the rational selection of targets for vaccine development. This approach has identified specific gene products of pathogens as vaccine targets. The expression and evaluation of these immunogens have led to the development of a successful vaccine for group B meningococcal strains through a process known as reverse vaccinology.25

Immune Biomarkers of Protection

The human immune response has been analyzed with sensitive high-throughput technologies that allow for systems biologic analysis of gene-expression patterns in lymphocytes and in microbes. Such information not only identifies susceptible microbial targets but also has the potential to define new biomarkers of protective immune responses, termed systems vaccinology.26 Mechanisms of protection and correlates of immunity can be rigorously explored in relevant animal models, but these properties can be definitively established in humans only through clinical trials and postlicensure surveillance. Such information enables precise immune activation, minimizes unintended side effects, and maximizes clinical efficacy. Successful protection may require neutralizing antibodies,18 effective T-cell responses,27 or possibly a combination of the two.

Dendritic Cells and Adjuvants

Figure 4. Figure 4. The Spectrum of Costimulation from Adjuvants to Viruses. A cellular and molecular understanding of dendritic-cell biology has facilitated improvements in vaccine-induced immune responses. Rather than generating responses through infection, immune stimulation can be achieved by increasingly complex modes of antigen presentation that range from introduction of selected proteins, with or without adjuvants, to gene-delivered immunogens, viruslike particles (VLP), structured arrays, or attenuated viruses. These approaches represent a spectrum of complexity and mimicry that elicits protective immunity without inflicting the adverse consequences of natural infection. HBV denotes hepatitis B virus, HPV human papillomavirus, VEE Venezuelan equine encephalitis, and WT wild type.

Critical to the modulation of the immune response is the presentation of specific antigens to the immune system. Dendritic cells play a central role in this process. Three subgroups of such cells, including two forms of myeloid dendritic cells and one plasmacytoid dendritic cell, each with distinct sets of toll receptors, modulate the response to specific antigens and adjuvants. Traditional vaccines have relied on live-attenuated or inactivated organisms, attenuated bacteria or capsules, or inactivated toxins.28,29 Progress has been made recently in enhancing immunity through a mechanistic understanding of the biology of dendritic cells and their response to adjuvants.30 Alternative delivery, including viruslike particles or structured arrays with the use of phage or nanoparticles, also stimulate effective immunity and provide powerful tools to confer protection for a specific pathogen (Figure 4).

Modes and Sites of Vaccine Delivery

An increasing number of vaccine vectors have become available to induce potent humoral or cellular immunity. Gene-based delivery of vaccine antigens effectively elicits immune responses by synthesizing proteins within antigen-presenting cells for endogenous presentation on major histocompatibility complex class I and II molecules. DNA-expression vectors, replication-defective viruses, or prime-boost combinations of the two31-35 have proved to be effective in eliciting broadly neutralizing antibodies, especially for influenza viruses.36,37

Prime-boost vaccine regimens that use DNA and viral vectors33 have increased both humoral immunity and memory CD8 T-cell responses.38 For example, a study of a vaccine regimen consisting of a poxvirus vector prime and protein boost (known as the RV144 trial) provided evidence that the vaccine prevented HIV-1 infection among persons in Thailand.39 Eliciting immune responses at portals of infection (e.g., in the respiratory and intestinal epithelial surfaces for pathogens such as influenza virus and rotavirus, respectively) may generate more efficient mucosal immunity. Similarly, waning vaccine responses require periodic boosting at defined times, requiring more integrated management of vaccines at all ages. Immunization in the elderly is of substantial concern because immune senescence can lead to a decrease in the responsiveness to vaccination.40