All eyes on the next generation of HIV vaccines: strategies for inducing a broadly neutralizing antibody response.

HIV-1 broadly neutralizing antibodies (BNAbs) develop after several years of infection through a recursive process of memory B cell adaptation and maturation against co-evolving virus quasispecies. Advances in single-cell sorting and memory B cell antibody cloning methods have identified many new HIV BNAbs targeting conserved epitopes on the HIV envelope (env) protein. 3D crystal structures and biophysical analyses of BNAbs bound to invariant virus structures expressed on monomeric gp120, epitope scaffolds, core structures, and native trimers have helped us to visualize unique binding interactions and paratope orientations that have been instrumental in guiding vaccine design. A paradigm shift in the approach to structure-based design of HIV-1 envelope immunogens came recently after several laboratories discovered that native viral envelopes or "env-structures" reverse-engineered to bind with high affinity to a handful of broadly neutralizing antibodies did not in fact bind the predicted germline precursors of these broadly neutralizing antibodies. A major challenge for HIV-1 B cell vaccine development moving forward is the design of new envelope immunogens that can trigger the selection and expansion of germline precursor and intermediate memory B cells to recapitulate B cell ontogenies associated with the maturation of a broadly neutralizing antibody response. Equally important for vaccine development is the identification of delivery systems, prime-boost strategies, and synergistic adjuvant combinations that can induce the magnitude and quality of antigen-specific T follicular helper (TFH) cell responses needed to drive somatic hypermutation (SHM) and B cell maturation against heterologous primary virus envelopes. Finding the combination of multi-protein envelope immunogens and immunization strategies that can evolve a potent broadly neutralizing antibody response portends to require a complex vaccine regimen that might be difficult to implement on any scale. This perspective strives to integrate recent insights into mechanisms associated with the evolution of an HIV-1 broadly neutralizing antibody response with current immunogen design and proffers a novel immunization strategy for skewing TH17/TFH cell responses that can drive B cell adaptation and affinity maturation associated with a broadly neutralizing antibody response.

Mucosal immunity and HIV-1 infection: applications for mucosal AIDS vaccine development.

Natural transmission of human immunodeficiency virus type 1 (HIV-1) occurs through gastrointestinal and vaginal mucosa. These mucosal tissues are major reservoirs for initial HIV replication and amplification, and the sites of rapid CD4(+) T cell depletion. In both HIV-infected humans and SIV-infected macaques, massive loss of CD4(+) CCR5(+) memory T cells occurs in the gut and vaginal mucosa within the first 10-14 days of infection. Induction of local HIV-specific immune responses by vaccines may facilitate effective control of HIV or SIV replication at these sites. Vaccines that induce mucosal responses, in particular CD8(+) cytotoxic T lymphocytes (CTL), have controlled viral replication at mucosal sites and curtailed systemic dissemination. Thus, there is strong justification for development of next generation vaccines that induce mucosal immune effectors against HIV-1 including CD8(+) CTL, CD4(+) T helper cells and secretory IgA. In addition, further understanding of local innate mechanisms that impact early viral replication will greatly inform future vaccine development. In this review, we examine the current knowledge concerning mucosal AIDS vaccine development. Moreover, we propose immunization strategies that may be able to elicit an effective immune response that can protect against AIDS as well as other mucosal infections.

Molecular pathways regulating CD4(+) T cell differentiation, anergy and memory with implications for vaccines.

CD4(+) T cells occupy a central role in the induction and regulation of adaptive immune responses. Activated CD4(+) T helper (Th) cells exert immediate effector functions by producing cytokines and chemokines, providing help for the induction of CD8(+) cytotoxic T lymphocyte responses and memory, and providing help for immunoglobulin class switching, affinity maturation of antibody and B cell memory. Inherent in naïve CD4(+) T cells is the flexibility to adopt alternate lineage potentials, which depend upon regulatory mechanisms that change with tissue microenvironment and upon infection. Here, we discuss lineage instructive programs that regulate CD4(+) T cell differentiation and memory and how to translate this knowledge into vaccines and immunotherapies that promote protective immune responses.

New paradigms for generating effective CD8+ T cell responses against HIV-1/AIDS.

CD8+ CTL responses are critical for eliminating virus infected cells in acute infection and in controlling virus replication during chronic infection. Despite evidence of potent HIV-1-specific CD8+ CTL responses during the earliest stage of acute infection leading to replacement of founder virus sequence(s) and resolution of peak viral load, in the majority of infected individuals, these responses are inadequate to prevent the establishment or control of persistent infection. Protective CD8+ CTL responses have yet to be achieved by vaccine approaches for HIV-1 or other viruses causing persistent infections, Mycobacterium tuberculosis, malaria, and cancer. Understanding the limitations of CD8+ CTL responses to keep pace with the diversity of rapidly evolving virus in the case of HIV-1 and HCV and to overcome the diverse and complex mechanisms persistent pathogens employ to escape immune recognition should lead to more effective prophylactic and therapeutic approaches for these diseases. Recent technological advances including single genome amplification (SGA) of plasma viral RNA along with direct amplicon sequencing to identify virus quasispecies, bioinformatics, and statistical methods for the systematic identification of HLA-class I associated escape mutations, and mathematical models that better define the kinetics of virus replication and decay, have provided significant insight into mechanisms of viral transmission and sequence evolution, virus-host interactions, and HIV-1 pathogenesis. In this review we attempt to integrate recent findings from studies in HIV-1, persistent virus infections, and cancer that predict effective T cell responses and suggest approaches that could shift the balance of control in favor of the host immune response. Here, we highlight factors considered essential for effective HIV-1 vaccine CD8+ T cell responses: vaccine antigens, quality, magnitude and breadth, mucosal targeting, and formation of CD8+ T cell mucosal memory.

Lessons learned from natural infection: focusing on the design of protective T cell vaccines for HIV/AIDS.

CD8(+) cytotoxic T lymphocyte (CTL) responses are crucial in establishing the control of persistent virus infections. Population studies of HIV-1-infected individuals suggest that CD8(+) CTL responses targeting epitopes that take the greatest toll on virus replication are instrumental in immune control. A major question for vaccine design is whether incorporating epitopes responsible for controlling a persistent virus will translate into protection from natural infection or serve solely as a fail-safe mechanism to prevent overt disease in infected individuals. Here, we discuss qualitative parameters of the CD8(+) CTL response and mechanisms operative in the control of persistent virus infections and suggest new strategies for design and delivery of HIV vaccines.

Memories that last forever: strategies for optimizing vaccine T-cell memory.

For acute self-limiting infections a vaccine is successful if it elicits memory at least as good as the natural experience; however, for persistent and chronic infections such as HIV, hepatitis C virus (HCV), human papillomavirus (HPV), and human herpes viruses, this paradigm is not applicable. At best, during persistent virus infection the person must be able to maintain the integrity of the immune system in equilibrium with controlling replicating virus. New vaccine strategies are required that elicit both potent high-avidity CD8(+) T-cell effector/memory and central memory responses that can clear the nidus of initial virus-infected cells at mucosal surfaces to prevent mucosal transmission or significantly curtail development of disease. The objective of an HIV-1 T-cell vaccine is to generate functional CD8(+) effector memory cells at mucosal portals of virus entry to prevent viral transmission. In addition, long-lived CD8(+) and CD4(+) central memory cells circulating through secondary lymphoid organs and resident in bone marrow, respectively, are needed to provide a concerted second wave of defense that can contain virus at mucosal surfaces and prevent systemic dissemination. Further understanding of factors which can influence long-lived effector and central memory cell differentiation will significantly contribute to development of effective T-cell vaccines. In this review we will focus on discussing mechanisms involved in T-cell memory and provide promising new approaches toward expanding current vaccine strategies to enhance antiviral memory.

What role does the route of immunization play in the generation of protective immunity against mucosal pathogens?

The route of vaccination is important in influencing immune responses at the initial site of pathogen invasion where protection is most effective. Immune responses required for mucosal protection can differ vastly depending on the individual pathogen. For some mucosal pathogens, including acute self-limiting infections, high-titer neutralizing Abs that enter tissue parenchyma or transude into the mucosal lumen are sufficient for clearing cell-free virus. However, for pathogens causing chronic infections such as HIV, hepatitis C virus, herpes viruses, mycobacteria, and fungal and parasitic infections, a single arm of the immune response generated by systemic vaccination may be insufficient for protection. Induction of the mucosal innate and adaptive immune systems, including CD4+ T help, Th17, high avidity CD8+ CTL, and secretory IgA and IgG1 neutralizing Abs, at the site of pathogen entry may be required for effective protection against highly invasive pathogens that lead to chronic infection and may be generated predominantly by mucosal vaccination.

Strategies for optimizing targeting and delivery of mucosal HIV vaccines.

Effective frontline defenses against HIV-1 will require targeting vaccines to mucosal tissue in order to induce alphabeta CD8(+) lymphocytes in mucosal effector sites (lamina propria and intraepithelial compartment) as well as antibody secreting plasma cells that can neutralize and limit free virus. A concerted second wave of assault against the virus will require the activation and recruitment of antigen specific memory CD4(+) and CD8(+) T cells in mesenteric lymph nodes and distal secondary lymphoid organs. New delivery strategies targeting the "right" DC subsets in combination with delivery of mucosal adjuvants and innate signals for activating DC will be essential for mucosal vaccines in order to circumvent the naturally tolerogenic environment and the induction of Tregs. Mucosal delivery of antigen in combination with inflammatory signals has been shown to empower systemic immunization by directing responses to mucosal sites for imprinting optimum mucosal memory. Here, we discuss novel vaccine strategies and adjuvants for optimizing mucosal delivery of HIV vaccines.

Strategies for recruiting and targeting dendritic cells for optimizing HIV vaccines.

The natural immune response against HIV and other pathogens that cause chronic infection is insufficient for protection. Novel vaccine design and delivery strategies for optimization of HIV vaccines are urgently needed. These will require a better understanding of a number of factors including: the interplay between dendritic cells (DCs) and multiple cell types in linking innate signals that orchestrate subsequent adaptive immune responses; the regulation of DC function by viral and bacterial vectors, adjuvants and immunomodulatory molecules; and the temporal and synergistic relationships between C-type lectins, Toll-like receptors, NOD-like receptors and RIG-1-like receptors, chemokines and cytokines in enhancing immune responses. Here, we discuss current vaccine strategies for optimizing the induction of immune responses by the recruitment of DCs and the targeting of vaccine antigens to DCs.

Functional CD8+ CTLs in mucosal sites and HIV infection: moving forward toward a mucosal AIDS vaccine.

The gastrointestinal and vaginal mucosa are the major routes of natural HIV transmission, with the former being the primary reservoir for virus replication. The crucial need for a global HIV vaccine underscores the effort to develop vaccines capable of eliciting mucosal immune responses. Vaccines that induce high levels of effective mucosal immunity should impact viral replication rate and prevent dissemination of virus from the mucosa into the systemic circulation. In our opinion, the major effort for effective HIV vaccine should be concentrated on generating protective immunity at the site of viral entry (i.e. the gastrointestinal and vaginal mucosae). Here we examine the current information regarding the role of mucosal immunity in prevention of HIV transmission and discuss strategies for mucosal AIDS vaccine development.

Generation of functionally active HIV-1 specific CD8+ CTL in intestinal mucosa following mucosal, systemic or mixed prime-boost immunization.

Gastrointestinal and vaginal mucosa are major sites of entry in natural HIV infection and therefore the preferred sites to elicit high-avidity CD8+ CTL by vaccination. We directly compare systemic and mucosal immunization in mice after DNA priming and boosting with rgp160 env expressed either in MVA or Ad for their ability to induce mucosal as well as systemic HIV-specific CTL. The optimal CTL response in the gut mucosa was observed after priming with the HIV-1 gp160 env DNA vaccine and boosting with rMVA or rAd encoding the same envelope gene all administered intrarectally (IR). Maximum levels of high-avidity CD8+ T cells were seen in intestinal lamina propria following this regimen. When the prime and boost routes were distinct, the delivery site of the boost had a greater impact than the DNA priming. IM DNA prime and IR rMVA boost were more effective than IR DNA prime and IM rMVA boost for eliciting mucosal CD8+ T-cell avidity. A systemic DNA-prime-followed by systemic rMVA boost induced high levels of high-avidity CD8+ T cells systemically, but responses were undetectable in mucosal sites. A single systemic immunization with rMVA was sufficient to induce high-avidity IFN-gamma secreting CD8+ T cells in systemic organs, whereas a single mucosal immunization with rMVA was not sufficient to elicit high-avidity CD8+ T cells in mucosa. Thus, a heterologous mucosal DNA prime-viral vectored boost strategy was needed. The requirement for a heterologous DNA prime-recombinant viral boost strategy for generation of high-avidity CD8+ T cells in mucosal sites in mice may be more stringent than for the induction of high-avidity CD8+ T cells in systemic compartments.

Role of alpha3 domain of class I MHC molecules in the activation of high- and low-avidity CD8+ CTLs.

CD8 can serve as a co-receptor or accessory molecule on the surface of CTL. As a co-receptor, CD8 can bind to the alpha3 domain of the same MHC class I molecules as the TCR to facilitate TCR signaling. To evaluate the role of the MHC class I molecule alpha3 domain in the activation of CD8(+) CTL, we have produced a soluble 227 mutant of H-2D(d), with a point mutation in the alpha3 domain (Glu227 --> Lys). 227 mutant class I-peptide complexes were not able to effectively activate H-2D(d)-restricted CD8 T cells in vitro, as measured by IFN-gamma production by an epitope-specific CD8(+) CTL line. However, the 227 mutant class I-peptide complexes in the presence of another MHC class I molecule (H-2K(b)) (that cannot present the peptide) with a normal alpha3 domain can induce the activation of CD8(+) CTL. Therefore, in order to activate CD8(+) CTL, the alpha3 domain of MHC class I does not have to be located on the same molecule with the alpha1 and alpha2 domains of MHC class I. A low-avidity CD8(+) CTL line was significantly less sensitive to stimulation by the 227 mutant class I-peptide complexes in the presence of the H-2K(b) molecule. Thus, low-avidity CTL may not be able to take advantage of the interaction between CD8 and the alpha3 domain of non-presenting class I MHC molecules, perhaps because of a shorter dwell time for the TCR-MHC interaction.

Progress on new vaccine strategies against chronic viral infections.

Among the most cost-effective strategies for preventing viral infections, vaccines have proven effective primarily against viruses causing acute, self-limited infections. For these it has been sufficient for the vaccine to mimic the natural virus. However, viruses causing chronic infection do not elicit an immune response sufficient to clear the infection and, as a result, vaccines for these viruses must elicit more effective responses--quantitative and qualitative--than does the natural virus. Here we examine the immunologic and virologic basis for vaccines against three such viruses, HIV, hepatitis C virus, and human papillomavirus, and review progress in clinical trials to date. We also explore novel strategies for increasing the immunogenicity and efficacy of vaccines.

Mucosal AIDS vaccines: current status and future directions.

Natural transmission of HIV occurs through mucosal surfaces. New information in immunology, virology and vaccinology has emerged regarding strategies for development of new mucosal vaccines against HIV. The intestinal mucosa represents a major site of HIV replication and amplification, and the initial site of CD4+ T-cell depletion. Local mucosal CD8+ cytotoxic T-lymphocytes (CTLs) and mucosal antibody can control AIDS virus replication within local tissues prior to systemic dissemination and can be more effective than a systemic immune response. Mucosal HIV vaccine delivery should be considered among the most effective immunization routes in the induction of mucosal antibody and CD8+ CTLs and protection against mucosal infection. New mucosal vaccine strategies, such as prime-boost, using a new generation of mucosal adjuvants, a synergistic combination of cytokines, chemokines, costimulatory molecules, CpG oligodeoxynucleotides, and targeting lymph nodes which drain mucosal sites, show promise to improve the efficacy of mucosal vaccines.

Progress on new vaccine strategies for the immunotherapy and prevention of cancer.

In recent years, great strides in understanding and regulating the immune system have led to new hope for harnessing its exquisite specificity to destroy cancer cells without affecting normal tissues. This review examines the fundamental immunologic advances and the novel vaccine strategies arising from these advances, as well as the early clinical trials studying new approaches to treat or prevent cancer.

Transcutaneous immunization induces mucosal CTLs and protective immunity by migration of primed skin dendritic cells.

Transcutaneous immunization (TCI), the application of vaccines on the skin, induces robust systemic and mucosal antibodies in animal models and in humans. The means by which mucosal immune responses to vaccine antigens are elicited by TCI has not been well characterized. We examined the effect of TCI with an HIV peptide vaccine on the induction of mucosal and systemic CTL responses and protective immunity against mucosal challenge with live virus in mice. Robust HIV-specific CTL responses in the spleen and in the gut mucosa were detected after TCI. The responses were dependent upon the addition of an adjuvant and resulted in protection against mucosal challenge with recombinant vaccinia virus encoding HIV gp160. Although it is clear that adjuvant-activated DCs migrated mainly to draining lymph nodes, coculture with specific T cells and flow cytometry studies with DCs isolated from Peyer's patches after TCI suggested that activated DCs carrying skin-derived antigen also migrated from the skin to immune-inductive sites in gut mucosa and presented antigen directly to resident lymphocytes. These results and previous clinical trial results support the observation that TCI is a safe and effective strategy for inducing strong mucosal antibody and CTL responses.

Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses.

The concern about bioterrorism with smallpox has raised the possibility of widespread vaccination, but the greater prevalence of immunocompromised individuals today requires a safer vaccine, and the mechanisms of protection are not well understood. Here we show that, at sufficient doses, the protection provided by both modified vaccinia Ankara and NYVAC replication-deficient vaccinia viruses, safe in immunocompromised animals, was equivalent to that of the licensed Wyeth vaccine strain against a pathogenic vaccinia virus intranasal challenge of mice. A similar variety and pattern of immune responses were involved in protection induced by modified vaccinia Ankara and Wyeth viruses. For both, antibody was essential to protect against disease, whereas neither effector CD4+ nor CD8+ T cells were necessary or sufficient. However, in the absence of antibody, T cells were necessary and sufficient for survival and recovery. Also, T cells played a greater role in control of sublethal infection in unimmunized animals. These properties, shared with the existing smallpox vaccine, provide a basis for further evaluation of these replication-deficient vaccinia viruses as safer vaccines against smallpox or against complications from vaccinia virus.

Cytokine, chemokine, and costimulatory molecule modulation to enhance efficacy of HIV vaccines.

Understanding key intervention points in developing immune responses may allow the rational inclusion of biological adjuvants into vaccines that could potentiate the immune response both quantitatively and qualitatively and enhance effective memory responses. Cytokine and chemokine combinations can potentially help target antigen to the appropriate antigen presenting cell and initiate maturation of these presenting cells, attract cells expressing different chemokine receptors, steer cellular immune responses toward Th1 and CD8 CTL, and enhance systemic and mucosal IgG and secretory IgA antibodies and determine their isotype balance. Animal protection studies suggest that synergistic combinations of cytokines and immunomodulating molecules may be required to protect from a viral challenge. For example, GM-CSF has been shown to be synergistic with IL-12 or CD40 ligand for induction of CTL and for antiviral protection, and the triple combination of GM-CSF, IL-12, and TNF alpha appears to induce the most effective protection in some mouse models. Chemokine-antigen fusions have also been shown to enhance immunogenicity of the antigen. Combinations of costimulatory molecules have been found to be synergistic when incorporated in a vaccine. Combined use of newer more potent vaccine constructs, containing codon optimized epitopes, relevant CpG motifs, cytokines, costimulatory molecules and chemokines, used in heterologous prime-boost strategies with viral vector vaccines or recombinant proteins, might afford the most potent vaccine approaches yet developed. In this review we will discuss the application and delivery of cytokines, costimulatory molecules, and chemokines toward improving current vaccine strategies.

Selective induction of high avidity CTL by altering the balance of signals from APC.

High avidity CTL are most effective at clearing viruses and cancer cells. Therefore, understanding the mechanisms involved in induction of high avidity CTL is critical for effective vaccines. However, no vaccine approach to selectively induce high avidity CTL in vivo has been discovered. In a new approach, signals from MHC class I (signal 1) and costimulatory molecules (signal 2) were adjusted by varying Ag dose and by use of recombinant poxvirus expressing a triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3), respectively. Independent of CTL avidity, a strong signal 1 resulted in an increased frequency of CD8(+) CTL. However, a strong signal 2 was necessary for the induction of high avidity CD8(+) CTL that killed target cells more efficiently, and signal 2 played a more crucial role in the absence of a strong signal 1. Only CTL induced with strong signal 2 killed tumor cells endogenously expressing low levels of Ag. Signal 2 contributed to the induction of high avidity CD8(+) CTL in both primary and secondary responses. Thus, although signal 2 has been known to increase the quantity of CTL response, in this study we show that it also improves the quality of CTL response. Our data also suggested that dendritic cells play an important role in induction of high avidity CD8(+) CTL in vivo. This strategy to selectively induce higher avidity CTL may lead to more effective vaccines for viruses and cancer.