Clofazimine for Treatment of Cryptosporidiosis in Human Immunodeficiency Virus Infected Adults: An Experimental Medicine, Randomized, Double-blind, Placebo-controlled Phase 2a Trial.

BACKGROUND: We evaluated the efficacy, pharmacokinetics (PK), and safety of clofazimine (CFZ) in patients living with human immunodeficiency virus (HIV) with cryptosporidiosis. METHODS: We performed a randomized, double-blind, placebo-controlled study. Primary outcomes in part A were reduction in Cryptosporidium shedding, safety, and PK. Primary analysis was according to protocol (ATP). Part B of the study compared CFZ PK in matched individuals living with HIV without cryptosporidiosis. RESULTS: Twenty part A and 10 part B participants completed the study ATP. Almost all part A participants had high viral loads and low CD4 counts, consistent with failure of antiretroviral (ARV) therapy. At study entry, the part A CFZ group had higher Cryptosporidium shedding, total stool weight, and more diarrheal episodes compared with the placebo group. Over the inpatient period, compared with those who received placebo, the CFZ group Cryptosporidium shedding increased by 2.17 log2 Cryptosporidium per gram stool (95% upper confidence limit, 3.82), total stool weight decreased by 45.3 g (P = .37), and number of diarrheal episodes increased by 2.32 (P = .87). The most frequent solicited adverse effects were diarrhea, abdominal pain, and malaise. One placebo and 3 CFZ participants died during the study. Plasma levels of CFZ in participants with cryptosporidiosis were 2-fold lower than in part B controls. CONCLUSIONS: Our findings do not support the efficacy of CFZ for the treatment of cryptosporidiosis in a severely immunocompromised HIV population. However, this trial demonstrates a pathway to assess the therapeutic potential of drugs for cryptosporidiosis treatment. Screening persons living with HIV for diarrhea, and especially Cryptosporidium infection, may identify those failing ARV therapy. CLINICAL TRIALS REGISTRATION: NCT03341767.

A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex.

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.

Mutagenesis of CXCR4 identifies important domains for human immunodeficiency virus type 1 X4 isolate envelope-mediated membrane fusion and virus entry and reveals cryptic coreceptor activity for R5 isolates.

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.

Both neutralization resistance and high infectivity phenotypes are caused by mutations of interacting residues in the human immunodeficiency virus type 1 gp41 leucine zipper and the gp120 receptor- and coreceptor-binding domains.

Neutralization resistance of human immunodeficiency virus type 1 (HIV-1) is a major impediment to vaccine development. We have found that residues of HIV-1 MN strain in the C terminus of gp120 and the leucine zipper (LZ) region of gp41 viral envelope proteins interact cooperatively to determine neutralization resistance and modulate infectivity. Further, results demonstrate that this interaction, by which regions of gp120 are assembled onto the LZ, involves amino acid residues intimately related to those which participate in the binding of the envelope to its receptor and coreceptor. Variations in this critical assembly structure determine the concordant, interdependent evolution of increased infectivity efficiency and neutralization resistance phenotypes of the envelopes. The results elucidate important structure-function relationships among epitopes that are important targets of vaccine development.

Primary virus envelope cross-reactivity of the broadening neutralizing antibody response during early chronic human immunodeficiency virus type 1 infection.

To test the hypothesis that changing neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1) during chronic infection were a response to emergence of neutralization escape mutants, we cloned expressed and characterized envelope clones from patients in the Multicenter AIDS Cohort Study (MACS). Pseudotyped HIV-1 envelope clones obtained from differing time points were assessed for sensitivity to neutralization by using sera from different times from the same and different patients. Clones from early and late time points during chronic infection had similar neutralization sensitivity, and neutralizing antibody responses cross-reacted with early, late, and heterologous envelopes. The potential for broadly effective HIV-1 immunization is supported.

Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response.

We have studied envelope protein from a donor with nonprogressive HIV-1 infection whose serum contains broadly cross-reactive, primary virus NA. DNA was extracted from lymphocytes, which had been collected approximately 6 and 12 months prior to the time of collection of the cross-reactive serum, and env genes were synthesized, cloned, expressed on pseudoviruses, and phenotyped in NA assays. Two clones from each time point had identical V3 region nucleotide sequences, utilized CCR5 but not CXCR4 for cell entry, and had similar reactivities with reference sera. Analysis of the full nucleotide sequence of one clone (R2) demonstrated it to be subtype B and have normal predicted glycosylation. R2 pseudovirus was compared with others expressing env genes of various clades for neutralization by sera from U.S. donors (presumed or known subtype B infections), and from individuals infected with subtypes A, C, D, E, and F viruses. Neutralization by the U.S. sera of R2 and other clade B pseudoviruses was low to moderate, although R2 was uniquely neutralized by all. R2 was neutralized by 3/3, 3/3, 2/5, 5/8, and 3/4 clade A, C, D, E, and F sera, respectively. R2 and a clade E pseudovirus were neutralized by largely complementary groups of sera, potentially defining two antigenic subgroups of HIV-1. The results suggest that the epitope(s) that induced the cross-clade reactive NA in donor 2 may be expressed on the R2 envelope.

Mutations in both gp120 and gp41 are responsible for the broad neutralization resistance of variant human immunodeficiency virus type 1 MN to antibodies directed at V3 and non-V3 epitopes.

The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative alpha-helix region of gp41. A series of chimeras from NS and NR env genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric env gene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.

Evolution of neutralizing antibody response against HIV type 1 virions and pseudovirions in multicenter AIDS cohort study participants.

Changes in neutralizing antibody (NA) titers in stored sera collected over 5 years from 10 participants in the Multicenter AIDS Cohort Study (MACS) were evaluated. The participants were HIV-1 infected on enrollment in the MACS, and remained AIDS free during the 5-year study interval. Seven viruses derived from molecular clones were used in NA assays; five of the viruses were T tropic (NL4-3, ALA1, NY5, SF2, and Z2Z6) and two were M tropic [AD8 and NL(SF162)]. In addition, pseudoviruses (PVs) were constructed that expressed envelope genes from NL4-3, ALA1, AD8, and SF162 and from primary viruses from two MACS participants (PV-9 and PV-10). There was significant correlation between NA titers obtained in four of five virus/PV comparisons, while the SF162 PV was more sensitive to NA than the corresponding virus. Comparable changes in NA titers were detected using viruses and PVs. Fourfold or greater increases in NA titers were noted in each of the participants, involving recognition of one to five of the nine strains tested. In some patients these NA titer changes appeared as discrete episodes of immune responses, while in others there may have been either multiple episodes or continuous evolution of the NA responses. The data indicate that changes in NA specificity occur during HIV-1 infection, which may result from the occurrence of neutralization escape mutation. The use of PVs for the study of phenotypic characteristics of envelope glycoproteins should facilitate the study of neutralization escape mutation in HIV-1 infection.

Annual influenza vaccination: immune response in patients over 10 years.

OBJECTIVE: To determine the effect of repeated annual influenza immunization on the host's serum antibody. DESIGN: Ten year observational study with cohort design. SETTING: Cystic Fibrosis Center at St. Vincent's Hospital and Medical Center, New York City, NY. PATIENTS: Thirty-eight children and young adults with cystic fibrosis (CF). MEASUREMENTS: Serum hemagglutination inhibition (HI) antibody titers were determined at the time of vaccination and 4 weeks later each year in the fall before the influenza epidemic. Shwachman scores were determined each year. RESULTS: While the pre-vaccination and post-vaccination geometric mean serum HI antibody titers varied from year to year, no upward or downward trend was evident over the 10 year period. The reciprocal of the post-vaccination geometric mean HI titers ranged annually from 32 to 74 for the influenza A (H3N2) vaccine strains, from 53 to 133 for the influenza A (H1N1) strains, and from 18 to 174 for influenza B strains. In addition, the majority of vaccinees had a presumably protective post-vaccination serum HI titer > or = 1:40 each year for all three vaccine strains. The initial mean Shwachman score of the group was 77. The final score of 76 after 10 years was not significantly different. CONCLUSIONS: Annual influenza vaccination appears to regularly induce presumably protective serum antibody levels in most CF children and young adults studied over a 10 year period.

Preparation and characterization of human HIV type 1 neutralizing reference sera.

Reference neutralizing antibody (NA) reagents are needed for laboratories to be able to compare results of neutralization assays that will be used to monitor HIV-1 vaccine recipients. In an effort to establish such reference reagents two asymptomatic, seropositive patients were identified with medium to high amounts of cross-reactive NA activity against a number of HIV-1 strains. Sera obtained from each individual at three or four sequential phlebotomies were pooled, and the two pools were each distributed in > 3000 aliquots into glass ampoules and lyophilized, and the ampoules were flame sealed. An HIV-1 antibody-negative reference serum was prepared in a similar fashion after pooling serum from four individuals. Ampoules were tested for uniformity of fill, sterility, moisture content, residual oxygen, stability, infectivity, and presence of antibody. An international collaborative study was conducted to determine the potency of the samples in six laboratories, each using their own neutralization assays and reagents. The results indicated reasonable consistency between laboratories and that both sera have sufficient titers against a variety of strains for use as reference reagents. These reference sera have been included in the World Health Organization (WHO) AIDS Reagent Project and are available through the three AIDS reagent repositories.

Variable infections with and serologic responses of rabbits to five different HIV-1 strains, including one neural tissue isolate.

The infectivity of different strains of HIV-1 in rabbits was investigated. The HIV-1RF and HIV-1MN inocula induced anti-envelope antibodies detectable by Western blot, and in the case of HIV-1RF, these antibodies were also detectable by ELISA. The peripheral blood lymphocytes (PBL) and lymph nodes from rabbits inoculated with HIV-1IIIB, HIV-1MN and HIV-1Z3, were positive for virus by culture and by polymerase chain reaction (PCR). HIV-1BRVA, originally isolated from a patient with AIDS dementia, infected the brain of the inoculated rabbit, as indicated by both virus culture and PCR. In this case PCR was positive using four different primer pairs. Throughout the study, rabbits showed no clinical signs of HIV-1 infection and no remarkable histopathology was observed in the tissues examined. The apparent differences in infectivity and tissue tropism of the five HIV-1 strains demonstrated here provide additional evidence that the rabbit may serve as a useful model for studying HIV-1 infection and pathogenesis.

High prevalence of antibodies to the gp120 V3 region principal neutralizing determinant of HIV-1MN in sera from Africa and the Americas.

Neutralizing antibodies (NA) against HIV-1MN and HIV-1IIIB, and antibodies binding to synthetic peptides (BA) derived from the gp120 envelope V3 region principal neutralizing determinants (PND) of the HIV-1MN, HIV-1IIIB, and HIV-1Z3 virus strains were assayed in HIV-1 antibody-positive sera from the United States, Haiti, Brazil, Zaire, and Zimbabwe. The ability of soluble PND peptide to block neutralization of the corresponding virus by representative sera was also tested. In each country, NA and BA titers were highest against the HIV-1MN strain, and compared with other countries, NA and BA titers against HIV-1MN were higher in sera from the United States and Haiti. When NA titers were compared with BA titers against either HIV-1MN or HIV-1IIIB, no correlation was found for the HIV-1IIIB strain, but there was a significant correlation for HIV-1MN. Addition of the HIV-1MN strain peptide to a neutralization assay for HIV-1MN resulted in a four- to tenfold reduction in NA titers in sera from the United States, Zaire, and Brazil. The results suggest that HIV-1MN and closely related variants are prevalent in many parts of the world, and that antibodies directed against the PND account for most of the neutralizing activity in sera of infected individuals.

PIP: Virologists assessed the extent of neutralizing antibody cross-reactivity to multiple virus strains in sera from 112 HIV-1 infected individuals from the US, Brazil, Haiti, Zaire, and Zimbabwe. They also looked at the association between virus neutralization and the level of antibody binding to synthetic peptides representing the HIV-1 gp120 V3 region principal neutralizing determinant (PND) sequences. The 3 strains observed included HIV-1 MN, HIV-1 Z3, and HIV-1 IIIB. Neutralizing antibodies (NA) and antibodies binding to synthetic peptides (BA) titers ranked highest against the PND sequence HIV-1 MN in all countries (p.01). These titers were higher in sera from the US and Haiti than sera from Brazil and Africa (p.05). A significant correlation existed between the NA and BA titers for HIV-1 MN (p.01), but not for HIV-1 IIIB. When the virologists added HIV-1 MN strain peptide to a neutralization assay for HIV-1 MN, NA titers in sera from the US, Zaire, and Brazil fell 4-10 fold. These findings intimated that HIV-1 MN and closely related variants are commonplace in several locations around the world, and that antibodies directed against HIV--1 gp120 V3 region PND sequences make up most of the neutralizing activity in sera of infected individuals. In conclusion, virologists need to conduct more studies that examine the true extent of strain variation worldwide. These studies could lay the groundwork for the development of an effective HIV-1 vaccine.

Lack of evidence for HIV-2 infection among at-risk individuals in Brazil.

Recent reports have indicated the possibility that HIV-2 has been introduced into groups at risk for AIDS in Brazil. We studied sera collected in 1987 and 1988 from 1,821 at-risk individuals from diverse regions in Brazil. Of the 1,821 sera, 367 (20%) were confirmed as being positive for HIV-1 antibodies by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), and Western blot. An additional 33 (2%) sera displayed some reactivity to HIV-2-infected cells by IF. All 33 sera were subsequently tested in HIV-1 and HIV-2 Western blots as well as an ELISA using HIV-1- or HIV-2-specific peptides. All sera were confirmed as positive for HIV-1 and negative for HIV-2 antibodies in both assays. We conclude that caution should be used in the interpretation of serologic cross-reactivity between HIV-1 and HIV-2 and that there is no evidence that HIV-2 has entered groups at risk for HIV infection in Brazil.

Effects of interferon-alpha in patients with AIDS-associated Kaposi's sarcoma are related to blood interferon levels and dose.

Definition of improved therapeutic regimens of interferon-alpha (IFN-alpha) for the treatment of Kaposi's sarcoma (KS) would be useful since currently recommended doses are sometimes associated with unacceptable toxicity. IFN concentrations were measured in serum samples from men with AIDS-associated KS who were enrolled in a trial of IFN-alpha alone (16 patients) or a trial of IFN-alpha combined with zidovudine (25 patients). Analyses were done to examine the relationship between the dose of IFN-alpha, blood level of IFN, and the patient's clinical response to treatment. There was no correlation between dose of zidovudine given and response. As expected, there was a high correlation between dose of IFN-alpha and blood level in both studies (p less than 0.001). Furthermore, we found relationships between clinical response and both dose of IFN-alpha and blood level achieved. In the two studies combined, among men with greater than 200 CD4+ cells/mm3 of blood at baseline on average daily doses of greater than or equal to 10 million international units (MIU) of IFN-alpha, 13/19 (68%) responded compared to 6/17 (35%) on less than MIU (p = 0.05). Similarly, of men with IFN blood levels greater than or equal to 100 IU/mL 12/16 (75%) responded compared to 7/20 (35%) of those with blood levels less than 100 IU/mL (p = 0.02). The dose and blood levels of IFN achieved and maintained may be important factors in determining responses of KS. Additional clinical trials of IFN-alpha treatment of KS at doses about 10 MIU/day appear warranted.

Synergistic effect of ganciclovir and foscarnet on cytomegalovirus replication in vitro.

Ganciclovir and foscarnet possess substantial activity against cytomegalovirus. Both exhibit dose-limiting toxicity, which reduces their clinical usefulness. We demonstrated synergistic inhibition of cytomegalovirus replication in vitro by ganciclovir and foscarnet. Reduced-dose combination therapy may provide a means to treat patients with cytomegalovirus infection while reducing drug toxicity.

Human immunodeficiency virus neutralizing antibodies in sera from North Americans and Africans.

Neutralizing antibodies (NAs) against four isolates of human immunodeficiency virus type 1 (HIV-1) were assayed in HIV-1 antibody positive sera from the United States, Haiti, Zimbabwe, and Zaire. Overall, there were NAs detected in 95, 81, 60, and 73% of sera with reciprocal geometric mean titers (GMTs) of 626, 23, 10, and 20, respectively, against HIV-1MN, HIV-1IIIB, HIV-1RF, and HIV-1Z3. Sera from North America had significantly higher NA titers against HIV-1MN. In each country, the highest antibody titers observed were against the MN strain. Otherwise, sera from the U.S. neutralized most strongly HIV-1IIIB, sera from Zaire neutralized most strongly HIV-1Z3, and sera from Zimbabwe had equal titers against all three viruses. The differences between countries were reflected in analyses of NA titers of subgroups classified on the basis of clinical status, indicating that the differences were not likely to be related to differences in clinical status of the patients being tested. Some of this antigenic variation is reflective of known genetic diversity, while some is not. The results suggest that undefined preserved and variable regions containing neutralization epitope(s) exist. These data do not indicate a need to define antigenic subtypes of HIV-1 at present. The existence of conserved neutralization epitope(s) may indicate the potential for broad immunogenicity of appropriately selected vaccine antigens.

Allogeneic bone marrow transplantation, zidovudine, and human immunodeficiency virus type 1 (HIV-1) infection. Studies in a patient with non-Hodgkin lymphoma.

Human immunodeficiency virus type 1 (HIV-1)-infected patients with non-Hodgkin lymphoma are classified as having the acquired immunodeficiency syndrome (AIDS). Allogeneic bone marrow transplantation is a successful therapy for patients with lymphoma who have a poor prognosis. Combined therapy with allogeneic bone marrow transplantation and the antiviral drug zidovudine has the potential advantage of protecting the new donor hematopoietic-lymphoid and monocyte-macrophage cells from HIV-1 infection. A 41-year-old man infected with HIV-1 who had lymphoma was treated with high-dose cyclophosphamide and total body irradiation followed by allogeneic bone marrow transplantation. Before transplantation he received high-dose zidovudine for 2 weeks (5 mg/kg body weight intravenously every 4 hours) and after transplantation he received a lower maintenance dose (1.33 mg/kg body weight intravenously every 4 hours). No untoward toxicities attributable to zidovudine were observed. Bone marrow engraftment occurred on day 17. Chromosome and restriction fragment length polymorphism analyses demonstrated complete chimerism. Peripheral blood mononuclear cells and bone marrow samples were negative for HIV-1 by culture and polymerase chain reaction gene amplification 32 days after transplantation. The patient died 47 days after transplantation because of tumor relapse. Analysis of autopsy tissue showed no evidence of HIV-1 by either culture (brain, bone marrow, lymph node, and tumor specimens) or by polymerase chain reaction gene amplification for HIV-1 RNA and DNA sequences (brain, bone marrow, heart, kidney, liver, lung, rectosigmoid, spleen, and tumor specimens). Immunologic monitoring showed loss of HIV-1 antibody. Adoptive immunologic transfer was shown to be present to both tetanus and diphtheria antigens. Our case suggests that the HIV-1-infected recipient cells may have been eradicated secondary to the bone marrow ablative chemo-radiotherapy and that zidovudine may be able to prevent the establishment of HIV-1 infection in donor hematopoietic-lymphoid cells.

Secretory immunoglobulin A antibody response is conserved in aged mice following oral immunization with influenza virus vaccine.

Parenteral immunization of BALB/c mice at 3 months of age with inactivated influenza virus vaccine elicited a haemagglutinin (HA)-specific serum IgG antibody response. The magnitude of this response declined with advancing age at the time of vaccination. By contrast, HA-specific IgA and IgG antibody levels observed in lung lavage fluids of mice immunized at 1 and 2 years of age were comparable to those of 5 month old mice when inactivated influenza virus vaccine was administered intragastrically. The secretory immune response was not fully developed in the first 3 weeks of life. However, the HA-specific IgA and IgG responses to oral vaccination in sera were reduced in 1 or 2 year old mice when compared to 5 month old mice. These data demonstrated the preservation of the virus-specific secretory IgA response in the pulmonary fluids of aged mice after oral vaccination with inactivated influenza virus vaccine. An age-dependent difference of systemic and mucosal immunity was evident in orally immunized mice.