Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 pg of purified DNA in saline, 67-95% of test mice and inoculations were undertaken (i) by a route that supports unusually efficient transfection (muscle), (ii) by routes that support less efficient transfection but represent routes frequently used for the administration of an antigen to a test animal (subcutaneous, intraperitoneal), and (iii) by routes that support less efficient transfection but deliver DNA to tissues with high levels of local immune surveillance (skin and respiratory passages).The effect of the route of inoculation on DNA vaccination was evaluated in murine and avian influenza virus models. In both models, the vaccine consisted of purified plasmid DNA that had been designed to express an influenza virus hemagglutinin glycoprotein. This glycoprotein mediates adsorption and penetration of virus and represents a major target for neutralizing antibody (16, 17). A number of antigenically distinct subtypes of hemagglutinin glycoproteins are found in naturally occurring influenza virus infections (18). In the murine model, plasmid DNA expressing the hemagglutinin subtype 1 (Hi) protein was used to protect against a lethal challenge with a mouse-adapted influenza virus with an identical Hi gene. In the chicken model, DNA expressing the hemagglutinin subtype 7 (H7) protein was used to vaccinate against a lethal H7 virus with an antigenically distinct H7 glycoprotein (19 §To whom reprint requests should be addressed.11478

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Virus-specific CD4 T cells are endowed with multiple functions, such as cytokine production, CD40 ligand (CD40L) expression (associated with the costimulation of CD8 and B cells), and degranulation (associated with cytotoxic potential). Here, we used antiviral CD4 T cells present in human blood to evaluate the relationship between cytokine production and other functions of CD4 T cells. Antiviral CD4 T cells specific for a virus causing persistent infection, cytomegalovirus (CMV), and two viruses causing nonpersistent infections, influenza virus and the smallpox vaccine virus (vaccinia virus), were studied. CD4 T cells specific for each of the viruses produced all seven possible combinations of the cytokines gamma interferon (IFN-␥), interleukin-2, and tumor necrosis factor alpha. Cells producing three or two cytokines (triple producers and double producers) represented nearly 50% of the total response to each of the viruses. Triple producers expressed the highest levels of cytokines per cell, and single producers expressed the lowest. Following stimulation, higher frequencies of triple producers than single producers expressed CD40L. Only CMV-specific CD4 T cells underwent degranulation. However, higher frequencies of CMV-specific triple producers than single producers showed this functional characteristic. In contrast to the functional phenotypes, the memory phenotypes of triple producers and IFN-␥ single producers did not differ. These results demonstrate a strong positive association between the cytokine coproduction capacity of a virus-specific CD4 T cell and its other functional characteristics and suggest that vaccines should aim to elicit T cells that coproduce more than one cytokine. Antiviral CD4 T cells play a vital role in the control of many viral infections. CD4 T cells are critical for the generation of functional CD8 T cells and neutralizing antibody. CD8 T-cell priming in the absence of CD4 help results in the generation of CD8 T cells that are defective in their ability to expand following a secondary antigen exposure (7,15,22,24). CD4 T-cell help is also critical for the maintenance of functional CD8 responses (25). Similarly, CD4 T cells are crucial for the entry of B cells into germinal center reaction sites for affinity maturation, a process that facilitates the generation of neutralizing antibody and the formation of memory B cells (10,17).Characteristics that are important for T-cell function in the control of viral infections include the production of cytokines, such as gamma interferon (IFN-␥), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-␣) (12, 23, 32), and proliferative capacity. Functional CD8 T-cell exhaustion is associated with a progressive loss of cytokine production and proliferative capacity, with the loss of IL-2 production being the first indicator of T-cell exhaustion, followed by the loss of TNF-␣ production (31). Apart from cytokine secretion, other indicators of CD4 T-cell function include costimulation via the expression of the CD40 ligand (CD40L) and cytot...

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Vaccination, or the deliberate induction of protective immunity by administering nonpathogenic forms of a microbe or its antigens to induce a memory immune response, is the world's most cost-effective medical procedure for preventing morbidity and mortality caused by infectious disease. Historically, most vaccines have worked by eliciting long-lived plasma cells. These cells produce antibodies that limit disease by neutralizing a toxin or blocking the spread of the infectious agent. For these 'B cell vaccines,' the immunological marker, or correlate, for protection is the titer of protective antibodies. With the discovery of HIV/AIDS, vaccine development has been confronted by an agent that is not easily blocked by antibody. To overcome this, researchers who are developing HIV/AIDS vaccines have turned to the elicitation of cellular immunity, or 'T cell vaccines,' which recognize and kill infected cells.

The ability of the C3d component of complement to enhance antibody responses and protective immunity to influenza virus challenges was evaluated using a DNA vaccine encoding a C3d fusion of the hemagglutinin (HA) from influenza virus. Plasmids were generated that encoded a transmembrane HA (tmHA), a secreted form of HA (sHA), or a sHA fused to three tandem copies of the murine homologue of the C3d (sHA-3C3d). Analysis of the titers, avidity maturation, and hemagglutinin-inhibition activity of raised antibody revealed that immunizations with sHA-3C3d DNA accelerated both the avidity maturation of antibody to HA and the appearance of hemagglutinin-inhibition activity. These accelerated antibody responses correlated to a more rapid appearance of protective immunity. They also correlated to complete protection from live virus challenge by a single vaccination at a dose ten times lower than the protective dose for non-C3d forms of HA.

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