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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.

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.

Mucosal tissues are major sites of HIV entry and initial infection. Induction of a local mucosal cytotoxic T lymphocyte response is considered an important goal in developing an effective HIV vaccine. In addition, activation and recruitment of memory CD4(+) and CD8(+) T cells in systemic lymphoid circulation to mucosal effector sites might provide the firewall needed to prevent virus spread. Therefore a vaccine that generates CD4(+) and CD8(+) responses in both mucosal and systemic tissues might be required for protection against HIV. However, optimal routes and number of vaccinations required for the generation of long lasting CD4(+) and CD8(+) CTL effector and memory responses are not well understood especially for mucosal T cells. A number of studies looking at protective immune responses against diverse mucosal pathogens have shown that mucosal vaccination is necessary to induce a compartmentalized immune response including maximum levels of mucosal high-avidity CD8(+) CTL, antigen specific mucosal antibodies titers (especially sIgA), as well as induction of innate anti-viral factors in mucosa tissue. Immune responses are detectable at mucosal sites after systemic delivery of vaccine, and prime boost regimens can amplify the magnitude of immune responses in mucosal sites and in systemic lymphoid tissues. We believe that the most optimal mucosal and systemic HIV/SIV specific protective immune responses and innate factors might best be achieved by simultaneous mucosal and systemic prime and boost vaccinations. Similar principals of vaccination may be applied for vaccine development against cancer and highly invasive pathogens that lead to chronic infection.

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., (Published by Elsevier Ltd.)

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.

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.

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.

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.

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.

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.

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.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

The ability of cytokines to steer CD4(+) T(h) cell responses toward a T(h)1 or T(h)2 phenotype and enhance the magnitude of both CD8(+) cytotoxic T lymphocytes (CTL) and antibody responses has clearly been demonstrated by our lab and others, but the influence of cytokines on protective immune responses is much less clear. Here we show an essential role for CD4(+) T(h)1 helper cell induction and IFN-gamma production in protection from viral challenge with a recombinant vaccinia virus expressing HIV-1MN viral envelope glycoprotein gp160. Complete protection from viral challenge is achieved only when the triple combination of exogenous cytokines granulocyte macrophage colony stimulating factor (GM-CSF), IL-12 and tumor necrosis factor (TNF)-alpha are co-administered with the peptide vaccine. In vivo depletion of CD4(+) cells or immunization of IFN-gamma-deficient mice abrogates protection. GM-CSF, IL-12 and TNF-alpha also synergize for the enhanced induction of CTL; however, adoptive transfer of a CD8(+) CTL line afforded only partial protection in this viral challenge model. As a possible mechanism of in vivo protection we show that GM-CSF increases the percentage and activity of antigen-presenting dendritic cells in draining lymph nodes where the immune response is initiated. We further demonstrate synergy between IL-12 and the proinflammatory cytokine TNF-alpha in driving IFN-gamma production. Thus, a combination of IL-12 and TNF-alpha is essential for the optimal development of T(h)1 responses and help for CTL induction in BALB/c mice, and is complemented by a third cytokine, GM-CSF, which enhances antigen presentation.

We used several approaches to develop enhanced vaccines for chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). 1) Selected epitopes were used to avoid potentially harmful immune responses. 2) Linkage between helper and cytotoxic T-lymphocyte (CTL) epitopes was found to be important. 3) We developed an "epitope enhancement" approach modifying the sequences of epitopes to make more potent vaccines, including examples for HIV and HCV epitopes presented by murine class II and human class I major histocompatibility complex (MHC) molecules. 4) CTL avidity was found to be important for clearing viral infections in vivo, and the mechanism was examined. High-avidity CTLs, however, were found to undergo apoptosis when confronted with high-density antigen, through a mechanism involving tumor necrosis factor (TNF), TNF-RII, and a permissive state induced through the T-cell receptor. 5) We employed cytokines in the adjuvant to steer immune responses toward desired phenotypes, and showed synergy between cytokines. 6) Intrarectal immunization with peptide vaccine induced mucosal and systemic CTL. Local mucosal CTL were found to be critical for resistance to mucosal viral transmission and this resistance was enhanced with mucosally delivered interleukin-12. 7) We used an asymmetry in induction of mucosal and systemic immune responses to circumvent pre-existing vaccinia immunity for use of recombinant vaccinia vaccines.

To improve the safety of recombinant vaccinia virus vaccines, modified vaccinia virus Ankara (MVA) has been employed, because it has a replication defect in most mammalian cells. Here we apply MVA to human immunodeficiency virus type 1 (HIV-1) vaccine development by incorporating the envelope protein gp160 of HIV-1 primary isolate strain 89.6 (MVA 89.6) and use it to induce mucosal cytotoxic-T-lymphocyte (CTL) immunity. In initial studies to define a dominant CTL epitope for HIV-1 89.6 gp160, we mapped the epitope to a sequence, IGPGRAFYAR (from the V3 loop), homologous to that recognized by HIV MN loop-specific CTL and showed that HIV-1 MN-specific CTLs cross-reactively recognize the corresponding epitope from strain 89.6 presented by H-2Dd. Having defined the CTL specificity, we immunized BALB/c mice intrarectally with recombinant MVA 89.6. A single mucosal immunization with MVA 89.6 was able to elicit long-lasting antigen-specific mucosal (Peyer's patch and lamina propria) and systemic (spleen) CTL responses as effective as or more effective than those of a replication-competent vaccinia virus expressing 89.6 gp160. Immunization with MVA 89.6 led to (i) the loading of antigen-presenting cells in vivo, as measured by the ex vivo active presentation of the P18-89.6 peptide to an antigen-specific CTL line, and (ii) the significant production of the proinflammatory cytokines (interleukin-6 and tumor necrosis factor alpha) in the mucosal sites. These results indicate that nonreplicating recombinant MVA may be at least as effective for mucosal immunization as replicating recombinant vaccinia virus.

Mucosal tissues are major sites of HIV entry and initial infection. Thus, the induction of a mucosal cytotoxic T lymphocyte (CTL) response is an important feature for an effective HIV vaccine. However, little is known about approaches to induce such a protective CTL response in the mucosa. Here for the first time we show that intrarectal immunization with a synthetic, multideterminant HIV peptide plus cholera toxin adjuvant induced long-lasting, antigen-specific CTL memory in both the inductive (Peyer's patch) and effector (lamina propria) mucosal sites, as well as in systemic sites (spleen), whereas systemic immunization induced specific CTL only in the spleen. Cholera toxin adjuvant, while enhancing the response, was not essential. The CTL recognized target cells either pulsed with HIV peptide or expressing endogenous whole envelope glycoprotein of Mr 160,000 (gp160). Exploring the requirements for CTL induction, we show that mucosal CTL responses are both interleukin 12 and interferon-gamma dependent by using antibody-treated and knock-out mice. Finally, to determine whether a mucosal response is actually protective against local mucosal challenge with virus, we show that intrarectal immunization with the synthetic HIV peptide vaccine protected mice against infection via mucosal challenge with a recombinant vaccinia virus expressing HIV-1IIIB gp160. These studies provide an approach to development of an HIV vaccine that induces CTL immunity in the mucosal and systemic immune systems and protects against mucosal infection with a virus expressing HIV-1 gp160.

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

To enhance and steer immune responses to synthetic peptide vaccines toward selected functional types and to understand the cooperative action of cytokines in fine-tuning the immune response, we attempted to influence the in vivo cytokine environment by delivering cytokines directly to the microenvironment in which the immune response is initiated. Here we study the effects of IL-2, IL-4, IL-7, IL-1beta, IL-12, IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) incorporated with peptide in adjuvant on a variety of responses elicited: CTL, T cell proliferation, cytokine production and message, and Ab isotype. We show GM-CSF to be the single most effective cytokine for enhancing both cellular and humoral immunity to two previously characterized HIV-1 MN vaccine constructs. Novel synergies were also detected. GM-CSF synergized with IL-12 for CTL induction in BALB/c mice concomitant with suppression of Th2 cytokines IL-4 and IL-10. TNF-alpha also synergized with IL-12, but by a different mechanism, inducing IFN-gamma production in BALB/c mice and thus shifting the response to a Th1 phenotype. The results presented here suggest that in addition to IL-2, optimum induction of CD8+ CTL in vivo requires a combination of cytokines, including GM-CSF (probably acting to enhance Ag presentation and CD4+ cell help) and IL-12 (steering the Th response toward Th1 cytokines).

Cytotoxic T lymphocytes and Th1 cells have been suggested to play a critical role in the control of HIV infection. It is therefore considered that a vaccine that induces a strong Th1 response and CTL response would be more efficacious than one that does not in providing protection against infection and progression toward AIDS. In this study we show that immunization with vaccine constructs consisting of multideterminant cluster peptides containing Th epitopes from the HIV-1IIIB envelope colinearly synthesized to peptide 18MN, is capable of inducing a Th1 response in mice and, dependent on this help, both cytotoxic T cell responses and neutralizing antibody toward the homologous strain of HIV. Moreover, the cytotoxic T cell response elicited by immunization with a mixture of cluster peptide-P18MN vaccine constructs was at least as cross-reactive against known viral variant P18 target sequences as a CTL line produced by immunization with a vaccinia construct expressing recombinant gp160 MN. Four adjuvants were compared to optimize both CTL and antibody responses. A single adjuvant formulation of peptide in ISA 51 could elicit all three: Th1 cells, CTLs, and neutralizing antibody. Thus, immunization directed toward the development of a cytotoxic T cell response does not preclude the development of neutralizing antibody and vice versa, i.e., the responses are not mutually exclusive. The immunization protocol described here should be directly applicable for study in clinical trials aimed at HIV-1 immunotherapy or prophylaxis.

Peptide constructs comprised of multideterminant Th peptides from the envelope glycoprotein of HIV previously identified to induce proliferative responses in four different haplotypes of mice and IL-2 responses in 52 to 73% of HIV positive, Ag-responsive patients, were colinearly synthesized with the peptide 18 of the V3 loop of HIV-1 gp 160, corresponding to the principal neutralizing determinant of HIV-IIIB. The segments containing clusters of overlapping Th epitopes were called cluster peptides. Cognate help for peptide 18 antibody was elicited after a single immunization in all strains of mice that had previously responded to a T cell epitope encompassed by the cluster peptides. Animals boosted with cluster peptide-peptide 18 constructs 36 to 52 wk later displayed secondary antibody responses. Cluster peptide 3-peptide 18 induced antibody that neutralized homologous virus in one strain of mice although strong peptide 18 antibody responses were detected in all four strains of mice. The most promising construct, cluster peptide 6-peptide 18, induced neutralizing antibody in all strains of mice tested, and in two strains the level of neutralizing antibody achieved was comparable to levels adequate for protection from homologous viral challenge in chimpanzees. After a single boost, antibody titers for 90% neutralization in the range of 1/1000 to 1/16,000 were achieved. These neutralizing titers against the homologous viral strain, after just two immunizations, are at least four- to eightfold higher than the highest titered other polyclonal V3-specific immune sera we have ever observed in our laboratories. We also asked why some sera neutralized and others with similar ELISA titers did not. No correlation was found between neutralization and isotype or affinity for peptide or gp 120. We could not account for neutralization by antibodies to the helper sites. Substitutions made in the central loop region of peptide 18, amino acid residues PGRAF, dramatically reduced binding of both neutralizing and non-neutralizing sera although some fine specificity differences between neutralizing and nonneutralizing sera were noted. These results have implications for the design of synthetic peptide vaccines for HIV.