Heather M. Kling

Persistent Pneumocystis Colonization Leads to the Development of Chronic Obstructive Pulmonary Disease in a Nonhuman Primate Model of AIDS

Human immunodeficiency virus (HIV)-infected patients are at increased risk for development of pulmonary complications, including chronic obstructive pulmonary disease (COPD). Inflammation associated with subclinical infection has been postulated to promote COPD. Persistence of Pneumocystis is associated with HIV infection and COPD, although a causal relationship has not been established. We used a simian/human immunodeficiency virus model of HIV infection to study pulmonary effects of Pneumocystis colonization. Simian/human immunodeficiency virus-infected/Pneumocystis-colonized monkeys developed progressive obstructive pulmonary disease characterized by increased emphysematous tissue and bronchial-associated lymphoid tissue. Increased levels of T helper type 2 cytokines and proinflammatory mediators in bronchoalveolar lavage fluid coincided with Pneumocystis colonization and a decline in pulmonary function. These results support the concept that an infectious agent contributes to the development of HIV-associated lung disease and suggest that Pneumocystis colonization may be a risk factor for the development of HIV-associated COPD. Furthermore, this model allows examination of early host responses important to disease progression, thus identifying potential therapeutic targets for COPD.

Pneumocystis colonization in immunocompetent and simian immunodeficiency virus-infected cynomolgus macaques.

Pneumocystis (Pc) colonization is common among human immunodeficiency virus (HIV)-infected subjects, although the clinical consequences of Pc carriage are not fully understood. We examined the frequency of asymptomatic carriage in healthy and simian immunodeficiency virus (SIV)-infected cynomolgus macaques by use of polymerase chain reaction (PCR) and assessment of changes in the serologic response to a recombinant fragment of the Pc protein kexin (KEX1). Anti-KEX1 antibodies were detected in 95% of healthy monkeys. To create a model of natural transmission of Pc, SIV-infected monkeys were cohoused with macaques coinfected with SIV and Pc. Pc colonization occurred when the CD4(+) T cell count decreased to <500 cells/microL, despite anti-Pc prophylaxis with trimethoprim-sulfamethoxazole. Increases in anti-KEX1 antibody titers preceded detection of Pc DNA in bronchoalveolar lavage (BAL) fluid samples by use of PCR. These results demonstrate the usefulness of recombinant KEX1 in serologic studies of Pc colonization and will improve the understanding of Pc transmission and clinical consequences of Pc colonization in HIV-infected patients.

Relationship of Pneumocystis jiroveci Humoral Immunity to Prevention of Colonization and Chronic Obstructive Pulmonary Disease in a Primate Model of HIV Infection

ABSTRACT Pulmonary colonization by the opportunistic pathogen Pneumocystis jiroveci is common in HIV+ subjects and has been associated with development of chronic obstructive pulmonary disease (COPD). Host and environmental factors associated with colonization susceptibility are undefined. Using a simian-human immunodeficiency virus (SHIV) model of HIV infection, the immunologic parameters associated with natural Pneumocystis jiroveci transmission were evaluated. SHIV-infected macaques were exposed to P. jiroveci by cohousing with immunosuppressed, P. jiroveci-colonized macaques in two independent experiments. Serial plasma and bronchoalveolar lavage (BAL) fluid samples were examined for changes in antibody titers to recombinant Pneumocystis-kexin protein (KEX1) and evidence of Pneumocystis colonization by nested PCR of BAL fluid. In experiment 1, 10 of 14 monkeys became Pneumocystis colonized (Pc+) by 8 weeks post-SHIV infection, while 4 animals remained Pneumocystis colonization negative (Pc−) throughout the study. In experiment 2, 11 of 17 animals became Pneumocystis colonized by 16 weeks post-SHIV infection, while 6 monkeys remained Pc−. Baseline plasma KEX1-IgG titers were significantly higher in monkeys that remained Pc−, compared to Pc+ monkeys, in experiments 1 (P = 0.013) and 2 (P = 0.022). Pc− monkeys had greater percentages of Pneumocystis-specific memory B cells after SHIV infection compared to Pc+ monkeys (P = 0.037). After SHIV infection, Pc+ monkeys developed progressive obstructive pulmonary disease, whereas Pc− monkeys maintained normal lung function throughout the study. These results demonstrate a correlation between the KEX1 humoral response and the prevention of Pneumocystis colonization and obstructive lung disease in the SHIV model. In addition, these results indicate that an effective Pneumocystis-specific memory B-cell response is maintained despite progressive loss of CD4+ T cells during SHIV infection.

Relationship of Pneumocystis Antibody Response to Severity of Chronic Obstructive Pulmonary Disease

Pneumocystis colonization has been associated with severity of chronic obstructive pulmonary disease (COPD). The relationship of Pneumocystis antibody status to COPD severity has not been investigated, but antibody levels might relate to both colonization susceptibility and COPD progression. We investigated anti-Pneumocystis antibody titers and airway obstruction in a cohort of patients with COPD. Undetectable anti-Pneumocystis antibody titer was an independent predictor of more-severe airway obstruction, although use of inhaled corticosteroids is a possible confounder of this effect.

Physiologic Changes in a Nonhuman Primate Model of HIV-Associated Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is increased in HIV, but its pathogenesis is not fully understood. Nonhuman primates infected with simian immunodeficiency virus (SIV) or SIV-HIV chimeric virus (SHIV) exhibit histologic changes characteristic of human PAH, but whether hemodynamic changes accompany this pathology is unknown. Repeated measurements of pulmonary artery pressures would permit longitudinal assessments of disease development and provide insights into pathogenesis. We tested the hypothesis that SIV-infected and SHIV-infected macaques develop physiologic manifestations of PAH. We performed right heart catheterizations, echocardiography, and computed tomography (CT) scans in macaques infected with either SIV (ΔB670) or SHIV (89.6P), and compared right heart and pulmonary artery pressures, as well as pulmonary vascular changes on CT scans, with those in uninfected control animals. Right atrial, right ventricular systolic, and pulmonary artery pressures (PAPs) were significantly elevated in 100% of macaques infected with either SIV or SHIV compared with control animals, with no difference in pulmonary capillary wedge pressure. PAPs increased as early as 3 months after SIV infection. Radiographic evidence of pulmonary vascular pruning was also found. Both SIV-infected and SHIV-infected macaques exhibited histologic changes in pulmonary arteries, predominantly consisting of intimal and medial hyperplasia. This report is the first to demonstrate SHIV-infected and SIV-infected macaques develop pulmonary hypertension at a high frequency, with physiologic changes occurring as early as 3 months after infection. These studies establish an important nonhuman primate model of HIV-associated PAH that will be useful in studies of disease pathogenesis and the efficacy of interventions.

Pulmonary vascular lesions are common in SIV- and SHIV-env-infected macaques.

The lack of animal models of HIV-related pulmonary arterial hypertension (HIV-PAH) severely limits investigation of this serious disease. While histological evidence of HIV-PAH has been demonstrated in macaques infected with simian immunodeficiency virus (SIV) as well as with chimeric simian/human immunodeficiency virus (SHIV) containing HIV-1-derived Nef protein, other primate models have not been studied. The objective was to document and describe the development of pulmonary vascular changes in macaques infected with SIV or with SIV containing HIV-1-derived envelope protein (SHIV-env). Lung tissue was obtained at necropsy from 13 SHIV (89.6P)-env-infected macaques and 10 SIV (ΔB670)-infected macaques. Pulmonary arterial pathology, including arterial hyperplasia and the presence of plexiform lesions, was compared to normal monkey lung. Pulmonary artery hyperplasia was present in 8 of 13 (62%) SHIV-env-infected macaques and 4/10 (36%) SIV-infected macaques. The most common histopathological lesions were intimal and medial hyperplasia of medium and large pulmonary arteries. Hyperplastic lesions were predominantly due to smooth muscle cell hyperplasia. This is the first report of pulmonary vascular lesions in SHIV-env-infected macaques and confirms prior reports of pulmonary vasculopathy in SIV-infected macaques. The finding of pulmonary arteriopathy in monkeys infected with SHIV not containing HIV-nef suggests that other factors might also be important in the development of HIV-PAH. This SHIV-env model provides a new means to investigate HIV-PAH.

Vaccine-Induced Immunogenicity and Protection Against Pneumocystis Pneumonia in a Nonhuman Primate Model of HIV and Pneumocystis Coinfection

BACKGROUND The ubiquitous opportunistic pathogen Pneumocystis jirovecii causes pneumonia in immunocompromised individuals, including human immunodeficiency virus (HIV)-infected individuals, and pulmonary colonization with P. jirovecii is believed to be a cofactor in the development of chronic obstructive pulmonary disease. There is no vaccine for P. jirovecii; however, most adults are seropositive, indicating natural immune priming to this pathogen. We have shown that humoral response to a recombinant subunit of the P. jirovecii protease kexin (KEX1) correlates with protection from P. jirovecii colonization and pneumonia. METHODS Here we evaluated the immunogenicity and protective capacity of the recombinant KEX1 peptide vaccine in a preclinical, nonhuman primate model of HIV-induced immunosuppression and Pneumocystis coinfection. RESULTS Immunization with KEX1 induced a robust humoral response remained at protective levels despite chronic simian immunodeficiency virus/HIV-induced immunosuppression. KEX1-immunized macaques were protected from Pneumocystis pneumonia, compared with mock-immunized animals (P= .047), following immunosuppression and subsequent natural, airborne exposure to Pneumocystis CONCLUSIONS These data support the concept that stimulation of preexisting immunological memory to Pneumocystis with a recombinant KEX1 vaccine prior to immunosuppression induces durable memory responses and protection in the context of chronic, complex immunosuppression.

Trimethoprim-sulfamethoxazole treatment does not reverse obstructive pulmonary changes in pneumocystis-colonized nonhuman primates with SHIV infection.

Background:Despite antiretroviral therapy and trimethoprim–sulfamethoxazole (TMP-SMX) prophylaxis, Pneumocystis pneumonia remains an important serious opportunistic infection in HIV-infected persons. Pneumocystis (Pc) colonization in HIV-infected individuals and in HIV-uninfected smokers is associated with chronic obstructive pulmonary disease (COPD). We previously developed a nonhuman primate model of HIV infection and Pc colonization and demonstrated that Pc colonization correlated with COPD development. In the present study, we examined kinetics of COPD development in non-human primate and tested the effect of Pc burden reduction on pulmonary function by TMP-SMX treatment. Methods:Cynomolgus macaques (n = 16) were infected with simian/human immunodeficiency virus (SHIV89.6P), and natural Pc colonization was examined by nested polymerase chain reaction of serial bronchoalveolar lavage fluid and anti-Pc serology. Results:Eleven of 16 monkeys became Pc colonized by 16 weeks post simian-human immunodeficiency virus (SHIV) infection. Pc colonization of SHIV-infected monkeys led to progressive declines in pulmonary function as early as 4 weeks after Pc detection. SHIV-infected and Pc-negative monkeys maintained normal lung function. At 25 weeks post-SHIV infection, TMP-SMX treatment was initiated in 7 Pc-positive (Pc+) (TMP: 20 mg/kg and SMX: 100 mg/kg, daily for 48 weeks) and 5 Pc-negative (Pc−) monkeys. Four SHIV+/Pc+ remained untreated for the duration of the experiment. Detection frequency of Pc in serial bronchoalveolar lavage fluid (P < 0.001), as well as plasma Pc antibody titers (P = 0.02) were significantly reduced in TMP-SMX–treated macaques compared with untreated. Conclusions:Reduction of Pc colonization by TMP-SMX treatment did not improve pulmonary function, supporting the concept that Pc colonization results in early, permanent obstructive changes in the lungs of immunosuppressed macaques.